Polypeptide Fragments Comprising Endonuclease Activity and Their Use

ABSTRACT

The present invention relates to polypeptide fragments comprising an amino-terminal fragment of the PA subunit of a viral RNA-dependent RNA polymerase or variants thereof possessing endonuclease activity, wherein said PA subunit is from a virus belonging to the Orthomyxoviridae family. This invention also relates to (i) crystals of the polypeptide fragments which are suitable for structure determination of said polypeptide fragments using X-ray crystallography and (ii) computational methods using the structural coordinates of said polypeptide to screen for and design compounds that modulate, preferably inhibit the endonucleolytically active site within the polypeptide fragment. In addition, this invention relates to methods identifying compounds that bind to the PA polypeptide fragments possessing endonuclease activity and preferably inhibit said endonucleolytic activity, preferably in a high throughput setting. This invention also relates to compounds and pharmaceutical compositions comprising the identified compounds for the treatment of disease conditions due to viral infections caused by viruses of the Orthomyxoviridae family.

TECHNICAL FIELD OF INVENTION

The present invention relates to polypeptide fragments comprising anamino-terminal fragment of the PA subunit of a viral RNA-dependent RNApolymerase or variants thereof possessing endonuclease activity, whereinsaid PA subunit is from a virus belonging to the Orthomyxoviridaefamily. This invention also relates to (i) crystals of the polypeptidefragments which are suitable for structure determination of saidpolypeptide fragments using X-ray crystallography and (ii) computationalmethods using the structural coordinates of said polypeptide to screenfor and design compounds that modulate, preferably inhibit theendonucleolytically active site within the polypeptide fragment. Inaddition, this invention relates to methods identifying compounds thatbind to the PA polypeptide fragments possessing endonuclease activityand preferably inhibit said endonucleolytic activity, preferably in ahigh throughput setting. This invention also relates to compounds andpharmaceutical compositions comprising the identified compounds for thetreatment of disease conditions due to viral infections caused byviruses of the Orthomyxoviridae family.

BACKGROUND OF THE INVENTION

Influenza is responsible for much morbidity and mortality in the worldand is considered by many as belonging to the most significant viralthreats to humans Annual Influenza epidemics swipe the globe andoccasional new virulent strains cause pandemics of great destructivepower. At present the primary means of controlling Influenza virusepidemics is vaccination. However, mutant Influenza viruses are rapidlygenerated which escape the effects of vaccination. In the light of thefact that it takes approximately 6 months to generate a new Influenzavaccine, alternative therapeutic means, i.e., antiviral medication, arerequired especially as the first line of defense against a rapidlyspreading pandemic.

An excellent starting point for the development of antiviral medicationis structural data of essential viral proteins. Thus, the crystalstructure determination of the Influenza virus surface antigenneuraminidase (von Itzstein et al., 1993, Nature 363:418-423) leddirectly to the development of neuraminidase inhibitors with anti-viralactivity preventing the release of virus from the cells, however, notthe virus production. These and their derivatives have subsequentlydeveloped into the anti-Influenza drugs, zanamivir (Glaxo) andoseltamivir (Roche), which are currently being stockpiled by manycountries as a first line of defense against an eventual pandemic.However, these medicaments provide only a reduction in the duration ofthe clinical disease. Alternatively, other anti-Influenza compounds suchas amantadine and rimantadine target an ion channel protein, i.e., theM2 protein, in the viral membrane interfering with the uncoating of thevirus inside the cell. However, they have not been extensively used dueto their side effects and the rapid development of resistant virusmutants (Magden et al., 2005, Appl. Microbiol. Biotechnol. 66:612-621).In addition, more unspecific viral drugs, such as ribavirin, have beenshown to work for treatment of Influenza infections (Eriksson et al.,1977, Antimicrob. Agents Chemother. 11:946-951). However, ribavirin isonly approved in a few countries, probably due to severe side effects(Furuta et al., 2005, Antimicrob. Agents Chemother. 49:981-986).Clearly, new antiviral compounds are needed, preferably directed againstdifferent targets.

Influenza virus A, B, C and Isavirus as well as Thogotovirus belong tothe family of Orthomyxoviridae which, as well as the family of theBunyaviridae, including the Hantavirus, Nairovirus, Orthobunyavirus,Phlebovirus, and Tospovirus, are negative stranded RNA viruses. Theirgenome is segmented and comes in ribonucleoprotein particles thatinclude the

RNA dependent RNA polymerase which carries out (i) the initial copyingof the single-stranded virion RNA (vRNA) into viral mRNAs and (ii) thevRNA replication. For the generation of viral mRNA the polymerase makesuse of the so called “cap-snatching” mechanism (Plotch et al., 1981,Cell 23:847-858; Kukkonen et al., 2005, Arch. Virol. 150:533-556; Leahyet al., 1997, J. Virol. 71:8347-8351; Noah and Krug, 2005, Adv. Virus

Res. 65:121-145). The polymerase is composed of three subunits: PB1(polymerase basic protein), PB2, and PA. For the cap-snatchingmechanism, the viral polymerase binds via its PB2 subunit to the 5′ RNAcap of cellular mRNA molecules which are cleaved at nucleotide 10 to 13by the endonucleolytic activity of the polymerase. The capped RNAfragments serve as primers for the synthesis of viral mRNAs by thenucleotidyl-transferase center in the PB 1 subunit (Li et al., 2001,EMBO J. 20:2078-2086). Finally, the viral mRNAs are 3′-endpolyadenylated by stuttering of the polymerase at an oligo-U motif atthe 5′-end of the template. Recent studies have precisely defined thestructural domain of PB2 responsible for capbinding (Fechter et al.,2003, J. Biol. Chem. 278:20381-20388; Guilligay et al., 2008 Nat.Struct. Mol. Biol. 15:500-506). The endonucleolytic activity of thepolymerase has hitherto been thought to reside in the PB1 subunit (Li etal, supra).

The polymerase complex seems to be an appropriate antiviral drug targetsince it is essential for synthesis of viral mRNA and viral replicationand contains several functional active sites likely to be significantlydifferent from those found in host cell proteins (Magden et al., supra).Thus, for example, there have been attempts to interfere with theassembly of polymerase subunits by a 25-amino-acid peptide resemblingthe PA-binding domain within PB1 (Ghanem et al., 2007, J. Virol.81:7801-7804). Moreover, there have been attempts to interfere withviral transcription by nucleoside analogs, such as2′-deoxy-2′-fluoroguanosine (Tisdale et al., 1995, Antimicrob. AgentsChemother. 39:2454-2458) and it has been shown that T-705, a substitutedpyrazine compound may function as a specific inhibitor of Influenzavirus RNA polymerase (Furuta et al., supra). Furthermore, theendonuclease activity of the polymerase has been targeted and a seriesof 4-substituted 2,4-dioxobutanoic acid compounds has been identified asselective inhibitors of this activity in Influenza viruses (Tomassini etal., 1994, Antimicrob. Agents Chemother. 38:2827-2837). In addition,flutimide, a substituted 2,6-diketopiperazine, identified in extracts ofDelitschia confertaspora, a fungal species, has been shown to inhibitthe endonuclease of Influenza virus (Tomassini et al., 1996, Antimicrob.Agents Chemother. 40:1189-1193). However, the inhibitory action ofcompounds on the endonucleolytic activity of the viral polymerase washitherto only studied in the context of the entire trimeric complex ofthe polymerase.

The PA subunit of the polymerase is functionally the leastwell-characterised, although it has been implicated in both cap-bindingand endonuclease activity, vRNA replication, and a controversialprotease activity. PA (716 residues in influenza A) is separable bytrypsination at residue 213. The recently determined crystal structureof the C-terminal two-thirds of PA bound to a PB 1 N-terminal peptideprovided the first structural insight into both a large part of the PAsubunit, whose function, however, still remains unclear, and the exactnature of one of the critical inter-subunit interactions (He et al.,2008, Nature 454:1123-1126; Obayashi et al., 2008, Nature454:1127-1131). Systematic mutation of conserved residues in the PAamino-terminal domain have identified residues important for proteinstability, promoter binding, cap-binding and endonuclease activity ofthe polymerase complex (Hara et al., 2006, J. Virol. 80:7789-7798). Theenzymology of the endonuclease within the context of intact viralribonucleoprotein particles (RNPs) has been extensively studied.

However, hitherto it was not possible to study the endonuclease activityof the PA subunit in the context of a polypeptide fragment possessingthe endonucleolytic activity, since it was not known which domain isresponsible for said activity. The present inventors surprisingly foundthat, contrary to the general opinion in the field, the endonucleolyticactivity resides exclusively within the amino-terminal region of the PAsubunit. The inventors have achieved to structurally characterize saiddomain by X-ray crystallography and identified the endonucleolyticactive center within the amino-terminal PA polypeptide fragment.

Thus, the present invention provides the unique opportunity to study theendonucleolytic activity of the viral polymerase in the context of apolypeptide fragment which will considerably simplify the development ofnew anti-viral compounds targeting the endonuclease activity of theviral polymerase as well as the optimization of previously identifiedcompounds. The surprising achievement of the present inventors torecombinantly produce PA polypeptide fragments possessing theendonucleolytic activity of the viral polymerase allows for performingin vitro high-throughput screening for inhibitors of a functional siteon the viral polymerase using easily obtainable material from astraightforward expression system. Furthermore, the structural data ofthe endonucleolytic PA polypeptide fragment as well as of theenzymatically active center therein allows for directed design ofinhibitors and in silico screening for potentially therapeuticcompounds.

It is an object of the present invention to provide (i) high resolutionstructural data of the endonucleolytic amino-terminal domain of theviral polymerase PA subunit by X-ray crystallography, (ii) computationalas well as in vitro methods, preferably in a high-throughput setting,for identifying compounds that can modulate, preferably inhibit, theendonuclease activity of the viral polymerase, preferably by blockingthe endonucleolytic active site within the PA subunit, and (iii)pharmacological compositions comprising such compounds for the treatmentof infectious diseases caused by viruses using the cap snatchingmechanism for synthesis of viral mRNA.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a polypeptidefragment comprising an amino-terminal fragment of the PA subunit of aviral RNA-dependent RNA polymerase possessing endonuclease activity,wherein said PA subunit is from a virus belonging to theOrthomyxoviridae family.

In a further aspect, the present invention relates to an isolatedpolynucleotide encoding an isolated polypeptide fragment according tothe present invention.

In a further aspect, the present invention relates to recombinant vectorcomprising the isolated polynucleotide according to the presentinvention.

In a further aspect, the present invention relates to a recombinant hostcell comprising the isolated polynucleotide according to the inventionor the recombinant vector according to the present invention.

In a further aspect, the present invention relates to a method foridentifying compounds which modulate the endonuclease activity of the PAsubunit of a viral RNA-dependent RNA polymerase from theOrthomyxoviridae family, comprising the steps of

(a) constructing a computer model of the active site defined by thestructure coordinates of the polypeptide fragment according to thepresent invention as shown in FIG. 18;(b) selecting a potential modulating compound by a method selected fromthe group consisting of:

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

(c) employing computational means to perform a fitting program operationbetween computer models of the said compound and the said active site inorder to provide an energy-minimized configuration of the said compoundin the active site; and(d) evaluating the results of said fitting operation to quantify theassociation between the said compound and the active site model, wherebyevaluating the ability of said compound to associate with the saidactive site.

In a further aspect, the present invention relates to a compoundidentifiable by the method according to the present invention, whereinsaid compound is able to modulate, preferably inhibit the endonucleaseactivity of the PA subunit or variant thereof

In a further aspect, the present invention relates to a method foridentifying compounds which modulate the endonuclease activity of the PAsubunit or polypeptide variants thereof, comprising the steps of (i)contacting the polypeptide fragment according to the invention or therecombinant host cell according to the invention with a test compoundand (ii) analyzing the ability of said test compound to modulate theendonuclease activity of said PA subunit polypeptide fragment.

In a further aspect, the present invention relates to a pharmaceuticalcomposition producible according to the in vitro method of the presentinvention.

In a further aspect, the present invention relates to a compoundidentifiable by the in vitro method according to the invention, whereinsaid compound is able to modulate, preferably inhibit the endonucleaseactivity of the PA subunit or variant thereof

In a further aspect, the present invention relates to an antibodydirected against the active site of the PA subunit or variant thereof

In a futher aspect, the present invention relates to the use of acompound according to the present invention, a pharmaceuticalcomposition according to the present invention, or an antibody accordingto the present invention for the manufacture of a medicament fortreating, ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Assay of thermal stability of the PA-Nter structure usingThermofluor. The thermal shift assay was performed with different metalions. For clarity, only the results obtained in absence of metal ion(full black line) or in presence of 1 mM MnCl₂ (dashed line) are shown.Arrows indicate the apparent melting temperature Tm.

FIG. 2: Effects of different metal ions on thermal stability of PA-Nter.Summary of the different melting points (Tm) extracted from the thermalshift assay at pH 8.0 with different metal ions. The effect of CoCl₂ onprotein stability at pH 7.0 was investigated but not interpretable dueto quenching by the metal.

FIG. 3: Effect of manganese on the structure of PA-Nter observed by farUV CD spectra. The secondary structure content of PA-Nter was monitoredin absence (full line) or presence of 1 mM MnCl₂ (dashed line).

FIG. 4: Assay of thermal stability with 2,4-Dioxo-4-phenylbutanoic acid(DPBA). Thermal shift assay with different concentrations of DPBA. DPBAfurther stabilizes PA-Nter in the presence of MnCl₂.

FIG. 5: Time series of the endonuclease activity of PA-Nter. 10 μMpurified panhandle RNA (ph-RNA) was incubated with 13 μM PA-Nter plus 1mM MnCl₂. The incubation at 37° C. was stopped by adding 20 mM EGTAafter 5, 10, 20, 40, and 80 minutes (lanes 4 to 8, respectively). Ascontrols, ph-RNA was incubated for 80 minutes at 37° C. with onlyPA-Nter (lane 1) only MnCl₂ (lane 2) or PA-Nter and MnCl₂ plus 20 mMEGTA. The reaction products were loaded on an 8% acrylamide/8 M urea geland stained with methylene blue.

FIG. 6: Effect of divalent cations on PA-Nter endonuclease (RNase)activity. In the top panel (A), purified ph-RNA plus PA-Nter wereincubated at pH 8 in the presence of β-mercaptoethanol and 1.5 mM MnCl₂,CaCl₂, MgCl₂, ZnCl₂, or CoCl₂. In the bottom panel (B), ph-RNA andPA-Nter were incubated at pH 7 with 1.5 mM MnCl₂, CaCl₂, MgCl₂, NiCl₂,or CoCl₂. After 30 minutes the reactions were stopped by adding 20 mMEGTA. Controls were performed using either salts or PA-Nter alone asindicated. The reaction products were loaded on 8% or 15% (for bottompanel) acrylamide/8 M urea and stained with methylene blue. Note that atpH 7, CoCl₂ stimulated the endonuclease stronger than MnCl₂. At pH 8,CoCl₂ precipitates and, thus, does not activate the endonucleaseactivity.

FIG. 7: PA-Nter endonuclease (RNase) activity on different RNAsubstrates. SRP Alu-RNA, tRNA, U-rich RNA, ph-RNA or short ph-RNA wereincubated with PA-Nter plus 1 mM MnCl₂ (lanes 2, 4, 6, 8, and 10) or inthe absence of PA-Nter (lanes 1, 3, 5, 7, and 9).

The digestion was performed at 37° C. After 40 minutes the reaction wasstopped by adding 20 mM EGTA. The reaction products were loaded on a 15%acrylamide/8 M urea gel and stained with methylene blue.

FIG. 8: Endonuclease activity of PA-Nter on single stranded DNA. Singlestranded DNA plasmid Ml3mp18 (100 ng/μl) (Fermentas) was incubated for60 minutes at 37° C. in the presence of PA-Nter plus MnCl₂ (lane 4). Thereaction was stopped by adding 20 mM EGTA. As controls, M13mp18 wasincubated with 1 mM MnCl₂ only (lane 2) or PA-Nter plus MnCl₂ and 20 mMEGTA (lane 3). The reaction products were loaded on a 0.8% agarose geland stained with ethidium bromide.

FIG. 9: Inhibition of PA-Nter endonuclease activity by2,4-Dioxo-4-phenylbutanoic acid (DPBA). Cleavage of ph-RNA (A) orM13mp18 ssDNA (B) by PA-Nter was tested at 37° C. during 40 minutes inthe presence of 1 mM MnCl₂ and increasing concentrations of DPBA (0,6.5, 13, 20, 26, 40, 65, 130, and 1000 μM). As a control, ph-RNA orssDNA was incubated with 1 mM MnCl₂ alone (lanes 1). The reactionproducts were loaded on 8% acrylamide/8 M urea and stained withmethylene blue (A) or on a 0.8% agarose gel and stained with ethidiumbromide (B).

FIG. 10: Three-dimensional structure of PA-Nter. Ribbon diagram of thestructure of influenza PA-Nter with a-helices (medium grey) andβ-strands (light grey). The key active site residues are indicated instick representation.

FIG. 11: Sequence alignment of polypeptide fragments derived from thePA-subunit of representative influenza strains: A/Victoria/3/1975 (humanH3N2; amino acid residues 1 to 209 of SEQ ID NO: 2),A/Duck/Vietman/1/2007 (avian H5N1; amino acid residues 1 to 209 of SEQID NO: 8), B/Ann Arbor/1/1966 (amino acid residues 1 to 206 of SEQ IDNO: 4) and C/Johannesburg/1/1966 (amino acid residues 1 to 189 of SEQ IDNO: 6). The secondary structure of A/Victoria/3/1975 is shown over thesequence alignment. The boxed sequences indicate sequence similaritybetween the four sequences. Residues in a solid black background areidentical between the four sequences. The triangles indicate the keyactive site residues.

FIG. 12: Representation of PA-Nter shaded according to residueconservation as based on the sequence alignment shown in FIG. 11, withgrey (not conserved), grey (equivalent residues) and black (100%conserved).

FIG. 13: Electrostatic surface potential of PA-Nter. The orientation isas in FIG. 12. Electrostatic surface potential of PA-Nter in the absenceof metal ions. The potential scale ranges from −10.0 kT/e (medium grey,acidic residues Asp(D) and Glu(E)) to 3.0 kT/e (dark grey, basicresidues Lys(K) and Arg(R)).

FIG. 14: Comparison of PA-Nter with other nucleases of the PD-(D/E)XKsuperfamily. Comparison of PA-Nter (left, A), P. furiosus Hollidayjunction resolvase Hjc (PDB entry 1 GEF) (middle, B) and E. coli EcoRVrestriction enzyme (PDB entry 1 STX, product complex with DNA andmanganese) (right, C) after superposition of the conserved core activesite structural motif. The rootmean-square-deviations are 2.9 Å for 77aligned Cα atoms of Hjc and 2.46 (3.1) Å for 55 (72) aligned Cα atoms ofEcoRV. Secondary-structure elements are as in FIG. 10 with key activesites residues in stick representation.

FIG. 15: Details of the manganese ion interactions with the active sitesof influenza

PA-Nter (molecule A) (left, A) and E. coli EcoRV restriction enzyme(product complex) (right, B). The active site elements and residues areshown respectively in leight grey and dark grey (left) and dark grey(right). Manganese ions and water molecules are respectively medium greyand dark grey spheres. The anomalous difference map contoured at 3 σ,calculated using manganese K edge (wavelength 1.89) diffraction data andmodel phases, is in dark grey. Peak heights are 14.1, 10.1, and 5.0 σfor Mn1, Mn2 and the sulphur of Cys45 respectively. Note that in metaldependent nucleases, the exact configuration of the metal ions andacidic side chains subtly depends on the reaction co-ordinate.

FIG. 16: Superposition of the active sites of influenza PA-Nter and E.coli EcoRV restriction enzyme. PA-Nter secondary structure elements andactive sites residues (indicated with PA) are shown in light grey withthe manganese ions in medim grey. Superposed are the equivalent elementsof EcoRV (PDB entry 1STX) (Horton and Perona, 2004, Biochemistry43:6841-6857) in dark grey (indicated with E) for the protein and darkgrey for the manganese ions. Key active site metal binding and catalyticfunctional groups of the two proteins align.

FIG. 17: Comparison of EcoRV product complex (B) and Pa-Nter with Glu66from a neighbouring molecule (A). The active site elements and residuesof PA-Nter (molecule A) are shown in light grey with manganese ions inmedium grey and the Glu66 containing loop of the adjacent molecule inlight grey. In the same orientation, after superposition of the twostructures, E. coli EcoRV restriction enzyme (PDB entry 1STX) (Hortonand Perona, supra) is shown in dark grey with the DNA bases in lightgrey and the manganese ions in medium grey. The carboxyl function ofGlu59 superimposes on the scissile phosphate of dA7 whereas thewell-ordered sulphate ion found in the active site of PA-Nter occupiesthe position of the phosphate part of dT8.

FIG. 18: Refined atomic structure coordinates for PA polypeptidefragment amino acids 1 to 209 according to amino acids 1 to 209 of theamino acid sequence set forth in SEQ ID NO: 2. There are three moleculesin the asymmetric unit denoted A, B, and C. The file header givesinformation about the structure refinement. “Atom” refers to the elementwhose coordinates are measured. The first letter in the column definesthe element. The 3-letter code of the respective amino acid is given andthe amino acid sequence position. The first 3 values in the line “Atom”define the atomic position of the element as measured. The fourth valuecorresponds to the occupancy and the fifth (last) value is thetemperature factor (B factor). The occupancy factor refers to thefraction of the molecules in which each atom occupies the positionspecified by the coordinates. A value of “1” indicates that each atomhas the same conformation, i.e., the same position, in all molecules ofthe crystal. B is a thermal factor that measures movement of the atomaround its atomic center. The anisotropic temperature factors are givenin the lines marked “ANISOU”. This nomenclature corresponds to the PDBfile format.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise. For example, if in a preferredembodiment the polypeptide fragment of the present invention correspondsto amino acids 1 to 209 of the amino acid sequence set forth in SEQ ID

NO: 2 and in another preferred embodiment the PA polypeptide fragmentaccording to the present invention may ba tagged with a peptide-tag thatis preferably cleavable from the PA polypeptide fragment, preferablyusing a TEV protease, it is a preferred embodiment of the invention thatthe polypeptide fragment corresponding to amino acids 1 to 209 of theamino acid sequence set forth in SEQ ID NO: 2 is tagged with apeptide-tag that is cleavable from the PA polypeptide using a TEVprotease.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, and recombinant DNAtechniques are employed which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. Asused in this specification and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

DEFINITIONS

The term “polypeptide fragment” refers to a part of a protein which iscomposed of a single amino acid chain. The term “protein” comprisespolypeptide fragments that resume a secondary and tertiary structure andadditionally refers to proteins that are made up of several amino acidchains, i.e., several subunits, forming quartenary structures. The term“peptide” refers to short amino acid chains of up to 50 amino acids thatdo not necessarily assume secondary or tertiary structures. A “peptoid”is a peptidomimetic that results from the oligomeric assembly ofN-substituted glycines.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. As is well known in the art, analogous positions in two ormore polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5. Those skilled in the art understand that it may benecessary to introduce gaps in either sequence to produce a satisfactoryalignment. For example, residues 1 to 196 in the Influenza A virus PAsubunit correspond to residues 1 to 195 and 1 to 178 in the Influenza Band C virus PA subunits, respectively. Residues in two or more PAsubunits are said to “correspond” if the residues are aligned in thebest sequence alignment. The “best sequence alignment” between twopolypeptides is defined as the alignment that produces the largestnumber of aligned identical residues. The “region of best sequencealignment” ends and, thus, determines the metes and bounds of the lengthof the comparison sequence for the purpose of the determination of thesimilarity score, if the sequence similarity, preferably identity,between two aligned sequences drops to less than 30%, preferably lessthan 20%, more preferably less than 10% over a length of 10, 20 or 30amino acids. A part of the best sequence alignment for the amino acidsequences of Influenza A (aa 1 to 209), B (aa 1 to 206), and C (aa 1 to189) PA subunits is shown in FIG. 11.

For example, amino acids Tyr24, His41, Glu80, Arg84, Leu106, Asp108,Glu119, Ile120, Tyr130, Glu133, Lys134, and Lys137 of the amino acidsequence set forth in SEQ ID NO: 2 (Influenza A virus PA subunit)correspond to amino acids Phe24, His41, Glu81, Arg85, Leu107, Asp109,Glu120, Val121, Tyr131, Lys134, Lys135, and Lys138 of the amino acidsequence set forth in SEQ ID NO: 4 (Influenza B virus PA subunit) andamino acids Ala24, His41, Glu65, Arg69, Leu91, Asp93, Glu104, Ile105,Tyr115, Ser118, Lys119, and Lys122 of the amino acid sequence set forthin SEQ ID NO: 6 (Influenza C virus PA subunit), respectively.

The present invention includes Influenza virus RNA-dependent RNApolymerase PA subunit fragments possessing endonuclease activity. Theterm “RNA-dependent RNA polymerase subunit PA” preferably refers to thePA subunit of Influenza A, Influenza B, or Influenza C virus, preferablyhaving an amino acid sequence as set out in SEQ ID NO: 2, 4, or 6.“RNA-dependent RNA polymerase subunit PA variants” have at least 60%,65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity,preferably sequence identity over the entire length of the fragmentusing the best sequence alignment and/or over the region of the bestsequence alignment, wherein the best sequence alignment is obtainablewith art known tools, e.g., Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap

Open 10.0, Gap Extend 0.5, with the amino acid sequence set forth in SEQID NO: 2, 4, or 6. It is preferred that when a naturally occurring PAvariant is aligned with a PA subunit according to SEQ ID NO: 2, 4, or 6that the alignment will be over the entire length of the two proteinsand, thus, that the alignment score will be determined on this basis. Itis, however, possible that the natural variant may compriseC-terminal/N-terminal or internal deletions or additions, e.g., throughN- or C-terminal fusions. In this case, only the best aligned region isused for the assessment of similarity and identity, respectively.Preferably and as set out in more detail below, fragments derived fromthese variants show the indicated similarity and identity, respectively,preferably within the region required for endonuclease activity.Accordingly, any alignment between SEQ ID NO: 2, 4, or 6 and a PAvariant should preferably comprise the endonuclease active site. Thus,the above sequence similarity and identity, respectively, to SEQ ID NO:2, 4, or 6 occurs at least over a length of 100, 110, 120, 130, 140,150, 160, 165, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300 or moreamino acids, preferably comprising the endonuclease active site. A largenumber of natural PA variants of sequences according to SEQ ID NO: 2, 4,or 6 are known and have been described in the literature. All these PAvariants are comprised and can be the basis for the polypeptidefragments of the present invention. Preferred examples of the InfluenzaA PA subunit, if SEQ ID NO: 2 is used as reference sequence, comprisemutations at one or more of positions Phe4, Ala20, Leu28, Glu31, Val44,Tyr48, Asn55, Gln57, Gly58, Val62, Leu65, Asp66, Thr85, Gly99, Ala100,Glu101, Ile118, Ile129, Asn142, Ile145, Glu154, Lys158, Asp164, Ile171,Lys172, Ile178, Asn184, and/or Arg204. In a preferred embodiment, saidvariant comprises one or more of the following mutations: Phe4Leu,Ala20Thr, Leu28Pro, Glu31Lys, Val44Ala, Tyr48His, Asn55Asp, Gln57Arg,Gly58Ser, Val62Ile, Leu65Ser, Asp66Gly, Thr85Ala, Gly99Lys, Ala100Val,Glu101Asp, Ile118Thr, Ile129Thr, Asn142Lys, Ile145Leu, Glu154Gly,Lys158Gln, Asp164Val, Ile171Val, Lys172Arg, Ile178Val, Asn184Ser,Asn184Arg, and/or Arg204Lys. Preferred variants of the Influenza B virusPA subunit, if SEQ ID NO: 4 is used as reference sequence, includemutations at one ore more of the following amino acid positions: Thr60,Asn86, Arg105, Asn158, His160, and/or Ile196. In a preferred embodimentthe Influenza B virus PA subunit variant comprises one or more of thefollowing mutations: Thr60Ala, Asn86Thr, Arg105Lys, Asn158Asp,His160Ser, and/or Ile196Val. Preferred variants of the Influenza C virusPA subunit, if SEQ ID NO: 6 is used as reference sequence, includemutations at one or more of the following amino acid positions: Thr11,Leu53, Ser58, Gly70, and/or Ala111. In a preferred embodiment, saidmutations are as follows: Thr11Ala, Leu53Met, Ser58Asn, Gly70Arg, and/orAla111Thr.

The polypeptide fragments of the present invention are, thus, based onRNA-dependent RNA polymerase subunit PA or variants thereof as definedabove. Accordingly, in the following specification the terms“polypeptide fragment(s)” and “PA polypeptide fragments” always comprisesuch fragments derived both from the PA proteins as set out in SEQ IDNO: 2, 4, or 6 and fragments derived from PA protein variants thereof,as set out above, possessing endonuclease activity. However, thespecification also uses the term “PA polypeptide fragment variants” or“PA fragment variants” to specifically refer to PA fragments possessingendonuclease activity that are derived from RNA-dependent RNA polymerasesubunit PA variants. The PA polypeptide fragments of the presentinvention thus preferably comprise, essentially consist or consist ofsequences of naturally occurring viral

PA subunits, preferably Influenza virus PA subunit. It is, however, alsoenvisioned that the PA fragment variants further contain amino acidsubstitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore amino acid positions, and have at least 60%, 65%, 70%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence similarity, preferably sequence identityover the entire length of the fragment using the best sequence alignmentand/or over the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with the amino acid sequence set forth inSEQ ID NO: 2, 4, or 6. It is understood that PA fragments of the presentinvention may comprise additional amino acids not derived from PA, like,e.g., tags, enzymes etc., such additional amino acids will not beconsidered in such an alignment, i.e., are excluded from the calculationof the alignment score. In a preferred embodiment, the above indicatedalignment score is obtained when aligning the sequence of the fragmentwith SEQ ID NO: 2, 4, or 6 at least over a length of 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 165, 170, 180, or 190 amino acids, whereinthe respective sequence of SEQ ID NO: 2, 4, or 6, preferably comprisesthe endonuclease active site.

In a preferred embodiment, the PA polypeptide fragment variants compriseat least the amino acid residues corresponding to amino acid residues 1to 196 of Influenza A virus PA or consist of amino acid residues 1 to196 (derived from SEQ ID NO: 2) and have at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence similarity, preferably sequence identity over theentire length of the fragment using the best sequence alignment and/orover the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g.,

Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5, with amino acid residues 1 to196 of the sequence set forth in SEQ ID NO: 2, more preferably the PApolypeptide fragment variants comprise at least the amino acid residuescorresponding to amino acid residues 1 to 209 of Influenza A virus PA orconsist of amino acid residues 1 to 209 (derived from SEQ ID NO: 2) andhave at least 70%, more preferably 75%, more preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% sequence similarity, preferably sequence identity over theentire length of the fragment using the best sequence alignment and/orover the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with the amino acid residues 1 to 209 ofthe amino acid sequence set forth in SEQ ID NO: 2, more preferably thePA polypeptide fragment variants comprise at least the amino acidresidues corresponding to amino acid residues 1 to 213 of Influenza Avirus PA or consist of amino acid residues 1 to 213 (derived from SEQ IDNO: 2) and have at least 60%, more preferably 65%, more preferably 70%,more preferably 75%, more preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequencesimilarity, preferably sequence identity over the entire length of thefragment using the best sequence alignment and/or over the region of thebest sequence alignment, wherein the best sequence alignment isobtainable with art known tools, e.g., Align, using standard settings,preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend0.5, with amino acid residues 1 to 213 of the amino acid sequence setforth in SEQ ID NO: 2. In preferred embodiments, the Influenza A virusPA polypeptide fragment variants of the present invention comprisemutations, preferably naturally occurring mutations such as mutations inone or more of the following amino acid residues when compared to SEQ IDNO: 2: Phe4, Ala20, Leu28, Glu31, Val44, Tyr48, Asn55, Gln57, Gly58,Val62, Leu65, Asp66, Thr85, Gly99, Ala100, Glu101, Ile118, Ile129,Asn142, Ile145, Glu154, Lys158, Asp164, Ile171, Lys172, Ile178, Asn184,and/or Arg204. In a preferred embodiment, said variant comprises one ormore of the following mutations: Phe4Leu, Ala20Thr, Leu28Pro, Glu31Lys,Val44Ala, Tyr48His, Asn55Asp, Gln57Arg, Gly58Ser, Val62Ile, Leu65Ser,Asp66Gly, Thr85Ala, Gly99Lys, Ala100Val, Glu101Asp, Ile118Thr,Ile129Thr, Asn142Lys, Ile145Leu, Glu154Gly, Lys 158Gln, Asp164Val,Ile171Val, Lys172Arg, Ile178Val, Asn184Ser, Asn184Arg, and/or Arg204Lys.

In a preferred embodiment, the PA polypeptide fragment variants compriseat least the amino acid residues corresponding to amino acid residues 1to 195 of Influenza B virus PA or consist of amino acid residues 1 to195 (derived from SEQ ID NO: 4) and have at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence similarity, preferably sequence identity over theentire length of the fragment using the best sequence alignment and/orover the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with amino acid residues 1 to 195 of theamino acid sequence set forth in SEQ ID NO: 4, more preferably the PApolypeptide fragment variants comprise at least the amino acid residuescorresponding to amino acid residues 1 to 206 of Influenza B virus PA orconsist of amino acid residues 1 to 206 (derived from SEQ ID NO: 4) andhave at least 70%, more preferably 75%, more preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% sequence similarity, preferably sequence identity over theentire length of the fragment using the best sequence alignment and/orover the region of the best sequence alignment, wherein the bestsequence alignment is obtainable with art known tools, e.g., Align,using standard settings, preferably EMBOSS::needle, Matrix: Blosum62,Gap Open 10.0, Gap Extend 0.5, with the amino acid residues 1 to 206 ofthe sequence set forth in SEQ ID NO: 4, more preferably the PApolypeptide fragment variants comprise at least the amino acid residuescorresponding to amino acid residues 1 to 210 of Influenza B virus PA orconsist of amino acid residues 1 to 210 (derived from SEQ ID NO: 4) andhave at least 60%, more preferably 65%, more preferably 70%, morepreferably 75%, more preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequencesimilarity, preferably sequence identity over the entire length of thefragment using the best sequence alignment and/or over the region of thebest sequence alignment, wherein the best sequence alignment isobtainable with art known tools, e.g., Align, using standard settings,preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend0.5, with amino acid residues 1 to 210 of the amino acid sequence setforth in SEQ ID NO: 4. In preferred embodiments, the Influenza B virusPA polypeptide fragment variants of the present invention comprisemutations, preferably naturally occurring mutations, at one ore more ofthe following amino acid positions compared to SEQ ID NO: 4: Thr60,Asn86, Arg105, Asn158, His160, and/or Ile196. In a preferred embodimentthe Influenza B virus PA subunit variant comprises one or more of thefollowing mutations: Thr60Ala, Asn86Thr, Arg105Lys, Asn158Asp,His160Ser, and/or Ile196Val.

In a preferred embodiment, the PA polypeptide fragment variants compriseat least the amino acid residues corresponding to amino acid residues 1to 178 of Influenza C virus PA or consist of amino acid residues 1 to178 (derived from SEQ ID NO: 6) and have at least 80%, more preferably85%, more preferably 90%, most preferably 95% sequence similarity overthe entire length of the fragment with amino acid residues 1 to 178 ofthe amino acid sequence set forth in SEQ ID NO: 6, more preferably thePA polypeptide fragment variants comprise at least the amino acidresidues corresponding to amino acid residues 1 to 189 of Influenza Cvirus PA or consist of amino acid residues 1 to 189 (derived from SEQ IDNO: 6) and have at least 70%, more preferably 75%, more preferably 80%,more preferably 85%, most preferably 90% sequence similarity over theentire length of the fragment with amino acid residues 1 to 189 of theamino acid sequence set forth in SEQ ID NO: 6, more preferably the PApolypeptide fragment variants comprise at least the amino acid residuescorresponding to amino acid residues 1 to 193 of Influenza C virus PA orconsist of amino acid residues 1 to 193 (derived from SEQ ID NO: 6) andhave at least 60%, more preferably 65%, more preferably 70%, morepreferably 75%, more preferably 80%, more preferably 85%, mostpreferably 90% sequence similarity over the entire length of thefragment with amino acid residues 1 to 193 of the amino acid sequenceset forth in SEQ ID NO: 6. In preferred embodiments, the Influenza Cvirus PA polypeptide fragment variants of the present invention comprisemutations, preferably naturally occurring mutations such as mutations inone or more of the following amino acid residues when compared to SEQ IDNO: 6: Thr11, Leu53, Ser58, Gly70, and/or Ala111. In a preferredembodiment, said mutations are as follows: Thr11Ala, Leu53Met, Ser58Asn,Gly70Arg, and/or Ala111Thr.

In the context of the present invention, the term “PA-Nter” refers to apolypeptide fragment which consists of amino acid residues 1 to 209 ofthe amino acid sequence as set forth in SEQ ID NO: 2 with an additionalamino-terminal linker, i.e., GMGSGMA (SEQ ID NO: 19).

If a PA polypeptide fragment of the present invention comprises one ofthe above outlined amino acid residues, it is preferred that the otheramino acid residues are not derived from the respective Influenza A, B,or C virus PA protein.

The term “sequence similarity” means that amino acids at the sameposition of the best sequence alignment are identical or similar,preferably identical. “Similar amino acids” possess similarcharacteristics, such as polarity, solubility, hydrophilicity,hydrophobicity, charge, or size. Similar amino acids are preferablyleucine, isoleucine, and valine; phenylalanine, tryptophan, andtyrosine; lysine, arginine, and histidine; glutamic acid and asparticacid; glycine, alanine, and serine; threonine, asparagine, glutamine,and methionine. The skilled person is well aware of sequence similaritysearching tools, e.g., available on the World Wide Web (e.g.,www.ebi.ac.uk/Tools/similarity.html).

The term “soluble”, as used herein, refers to a polypeptide fragmentwhich remains in the supernatant after centrifugation for 30 min at100,000×g in an aqueous buffer under physiologically isotonicconditions, for example, 0.14 M sodium chloride or sucrose, at a proteinconcentration of at least 200 μg/ml, preferably of at least 500 μg/ml,preferably of at least 1 mg/ml, more preferably of at least 2 mg/ml,even more preferably of at least 3 mg/ml, even more preferably of atleast 4 mg/ml, most preferably of at least 5 mg/ml in the absence ofdenaturants such as guanidine or urea in effective concentrations. Aprotein fragment that is tested for its solubility is preferablyexpressed in one of the cellular expression systems indicated below.

The term “purified” in reference to a polypeptide, does not requireabsolute purity such as a homogenous preparation, rather it representsan indication that the polypeptide is relatively purer than in thenatural environment. Generally, a purified polypeptide is substantiallyfree of other proteins, lipids, carbohydrates, or other materials withwhich it is naturally associated, preferably at a functionallysignificant level, for example, at least 85% pure, more preferably atleast 95% pure, most preferably at least 99% pure. The expression“purified to an extent to be suitable for crystallization” refers to aprotein that is 85% to 100%, preferably 90% to 100%, more preferably 95%to 100% pure and can be concentrated to higher than 3 mg/ml, preferablyhigher than 10 mg/ml, more preferably higher than 18 mg/ml withoutprecipitation. A skilled artisan can purify a polypeptide using standardtechniques for protein purification. A substantially pure polypeptidewill yield a single major band on a non-reducing polyacrylamide gel.

The term “associate” as used in the context of identifying compoundswith the methods of the present invention refers to a condition ofproximity between a moiety (i.e., chemical entity or compound orportions or fragments thereof), and an endonuclease active site of thePA subunit. The association may be non-covalent, i.e., where thejuxtaposition is energetically favored by, for example,hydrogen-bonding, van der Waals, electrostatic, or hydrophobicinteractions, or it may be covalent.

The term “endonuclease activity” or “endonucleolytic activity” refers toan enzymatic activity which results in the cleavage of thephosphodiester bond within a polynucleotide chain. In the context of thepresent invention, the polypeptide fragments possess an endonucleolyticactivity, which is preferably not selective for the polynucleotide type,i.e., the polypeptide fragments according to the present inventionpreferably exhibit endonucleolytic activity for DNA and RNA, preferablyfor single stranded DNA (ssDNA) or single stranded RNA (ssRNA). In thiscontext, “Single stranded” means that a stretch of preferably at least 3nucleotides, preferably at least 5 nucleotides, more preferably at least10 nucleotides within the polynucleotide chain are single stranded,i.e., not base paired to another nucleotide. Preferably, theendonucleolytic activity of the polypeptide fragments according to thepresent invention is not dependent on recognition sites, i.e., specificnucleotide sequences, but results in unspecific cleavage ofpolynucleotide chains. For example, the skilled person may test forendonucleolytic activity of polypeptide fragments according to thepresent invention by incubating RNA or DNA substrates such as panhandleRNA or a linear or circular single stranded DNA, e.g., the circularM13mp18 DNA (MBI Fermentas), with or without the respective polypeptidefragment, for example, at 37° C. for a certain period of time such asfor 5, 10, 20, 40, 60, or 80 minutes, and test for the integrity of thepolynucleotides, for example, by gel electrophoresis.

The term “nucleotide” as used herein refers to a compound consisting ofa purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g., as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992) and further include,but are not limited to, synthetic nucleosides having modified basemoieties and/or modified sugar moieties, e.g., described generally byScheit, Nucleotide Analogs (John Wiley, N.Y., 1980).

The term “isolated polynucleotide” refers to polynucleotides that were(i) isolated from their natural environment, (ii) amplified bypolymerase chain reaction, or (iii) wholly or partially synthesized, andmeans a single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases and includes DNA and RNA molecules, both sense andanti-sense strands. The term comprises cDNA, genomic DNA, andrecombinant DNA. A polynucleotide may consist of an entire gene, or aportion thereof.

The term “recombinant vector” as used herein includes any vectors knownto the skilled person including plasmid vectors, cosmid vectors, phagevectors such as lambda phage, viral vectors such as adenoviral orbaculoviral vectors, or artificial chromosome vectors such as bacterialartificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1artificial chromosomes (PAC). Said vectors include expression as well ascloning vectors. Expression vectors comprise plasmids as well as viralvectors and generally contain a desired coding sequence and appropriateDNA sequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments.

“Recombinant host cell”, as used herein, refers to a host cell thatcomprises a polynucleotide that codes for a polypeptide fragment ofinterest, i.e., the PA polypeptide fragment or variants thereofaccording to the invention. This polynucleotide may be found inside thehost cell (i) freely dispersed as such, (ii) incorporated in arecombinant vector, or (iii) integrated into the host cell genome ormitochondrial DNA. The recombinant cell can be used for expression of apolynucleotide of interest or for amplification of the polynucleotide orthe recombinant vector of the invention. The term “recombinant hostcell” includes the progeny of the original cell which has beentransformed, transfected, or infected with the polynucleotide or therecombinant vector of the invention. A recombinant host cell may be abacterial cell such as an E. coli cell, a yeast cell such asSaccharomyces cerevisiae or Pichia pastoris, a plant cell, an insectcell such as SF9 or Hi5 cells, or a mammalian cell. Preferred examplesof mammalian cells are Chinese hamster ovary (CHO) cells, green Africanmonkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELAcells, and the like.

As used herein, the term “crystal” or “crystalline” means a structure(such as a three-dimensional solid aggregate) in which the plane facesintersect at definite angles and in which there is a regular structure(such as internal structure) of the constituent chemical species. Theterm “crystal” can include any one of: a solid physical crystal formsuch as an experimentally prepared crystal, a crystal structurederivable from the crystal (including secondary and/or tertiary and/orquaternary structural elements), a 2D and/or 3D model based on thecrystal structure, a representation thereof such as a schematicrepresentation thereof or a diagrammatic representation thereof, or adata set thereof for a computer. In one aspect, the crystal is usable inX-ray crystallography techniques. Here, the crystals used can withstandexposure to X-ray beams and are used to produce diffraction pattern datanecessary to solve the X-ray crystallographic structure. A crystal maybe characterized as being capable of diffracting X-rays in a patterndefined by one of the crystal forms depicted in T. L. Blundell and L. N.Johnson, “Protein Crystallography”, Academic Press, New York (1976).

The term “unit cell” refers to a basic parallelepiped shaped block. Theentire volume of a crystal may be constructed by regular assembly ofsuch blocks. Each unit cell comprises a complete representation of theunit of pattern, the repetition of which builds up the crystal.

The term “space group” refers to the arrangement of symmetry elements ofa crystal. In a space group designation the capital letter indicates thelattice type and the other symbols represent symmetry operations thatcan be carried out on the contents of the asymmetric unit withoutchanging its appearance.

The term “structure coordinates” refers to a set of values that definethe position of one or more amino acid residues with reference to asystem of axes. The term refers to a data set that defines thethree-dimensional structure of a molecule or molecules (e.g., Cartesiancoordinates, temperature factors, and occupancies). Structuralcoordinates can be slightly modified and still render nearly identicalthree-dimensional structures. A measure of a unique set of structuralcoordinates is the root mean square deviation of the resultingstructure. Structural coordinates that render three-dimensionalstructures (in particular, a three-dimensional structure of anenzymatically active center) that deviate from one another by a rootmean square deviation of less than 3 Å, 2 Å, 1.5 Å, 1.0 Å, or 0.5 Å maybe viewed by a person of ordinary skill in the art as very similar.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a variant of the PA polypeptide fragment orthe enzymatically active center therein from the backbone of the PApolypeptide fragment or the enzymatically active center therein asdefined by the structure coordinates of the PA polypeptide fragmentPA-Nter according to FIG. 18.

As used herein, the term “constructing a computer model” includes thequantitative and qualitative analysis of molecular structure and/orfunction based on atomic structural information and interaction models.The term “modeling” includes conventional numeric-based moleculardynamic and energy minimization models, interactive computer graphicmodels, modified molecular mechanics models, distance geometry, andother structure-based constraint models.

The term “fitting program operation” refers to an operation thatutilizes the structure coordinates of a chemical entity, anenzymatically active center, a binding pocket, molecule or molecularcomplex, or portion thereof, to associate the chemical entity with theenzymatically active center, the binding pocket, molecule or molecularcomplex, or portion thereof. This may be achieved by positioning,rotating or translating the chemical entity in the enzymatically activecenter to match the shape and electrostatic complementarity of theenzymatically active center. Covalent interactions, non-covalentinteractions such as hydrogen bond, electrostatic, hydrophobic, van derWaals interactions, and non-complementary electrostatic interactionssuch as repulsive charge-charge, dipole-dipole and charge-dipoleinteractions may be optimized. Alternatively, one may minimize thedeformation energy of binding of the chemical entity to theenzymatically active center.

As used herein, the term “test compound” refers to an agent comprising acompound, molecule, or complex that is being tested for its ability toinhibit the endonucleolytic activity of the polypeptide fragment ofinterest, i.e., the PA polypeptide fragment of the invention or variantsthereof possessing endonucleolytic acitvity. Test compounds can be anyagents including, but not restricted to, peptides, peptoids,polypeptides, proteins (including antibodies), lipids, metals,nucleotides, nucleotide analogs, nucleosides, nucleic acids, smallorganic or inorganic molecules, chemical compounds, elements,saccharides, isotopes, carbohydrates, imaging agents, lipoproteins,glycoproteins, enzymes, analytical probes, polyamines, and combinationsand derivatives thereof The term “small molecules” refers to moleculesthat have a molecular weight between 50 and about 2,500 Daltons,preferably in the range of 200-800 Daltons. In addition, a test compoundaccording to the present invention may optionally comprise a detectablelabel. Such labels include, but are not limited to, enzymatic labels,radioisotope or radioactive compounds or elements, fluorescent compoundsor metals, chemiluminescent compounds and bioluminescent compounds. Wellknown methods may be used for attaching such a detectable label to atest compound. The test compound of the invention may also comprisecomplex mixtures of substances, such as extracts containing naturalproducts, or the products of mixed combinatorial syntheses. These canalso be tested and the component that inhibits the endonucleolyticactivity of the target polypeptide fragment can be purified from themixture in a subsequent step. Test compounds can be derived or selectedfrom libraries of synthetic or natural compounds. For instance,synthetic compound libraries are commercially available from MaybridgeChemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (SanDiego, Calif.), or Aldrich (Milwaukee, Wis.). A natural compound libraryis, for example, available from TimTec LLC (Newark, Del.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal cell and tissue extracts can be used.Additionally, test compounds can be synthetically produced usingcombinatorial chemistry either as individual compounds or as mixtures. Acollection of compounds made using combinatorial chemistry is referredto herein as a combinatorial library.

In the context of the present invention, “a compound which modulates theendonucleolytic activity” may increase or decrease, preferably inhibitthe endonucleolytic activity of the PA subunit or the viralRNA-dependent RNA polymerase or a variant thereof Preferably, such acompound is specific for the endonucleolytic activity of the viral PAsubunit or variant thereof and does not modulate, preferably decreasethe endonucleolytic activity of other endonucleases, in particularmammalian endonucleases.

The term “a compound which decreases the endonucleolytic activity” meansa compound which decreases the endonucleolytic activity of the PAsubunit of the viral RNA-dependent RNA polymerase from theOrthomyxoviridae family or a variant thereof by 50%, more preferably by60%, even more preferably by 70%, even more preferably by 80%, even morepreferably by 90%, and most preferably by 100% compared to theendonucleolytic activity of the PA subunit or a variant thereof withoutsaid compound but with otherwise the same reaction conditions, i.e.,buffer conditions, reaction time and temperature. It is most preferredthat the compound which decreases the endonucleolytic activity of the PAsubunit or a variant thereof inhibits said activity, i.e., decreasessaid activity by at least 95%, preferably by 100% compared to theactivity without the compound. It is particularly preferred that thecompound that decreases or inhibits the endonucleolytic activity of thePA subunit or a variant thereof specifically decreases or inhibits theendonucleolytic activity of the PA subunit or a variant thereof but doesnot inhibit the endonucleolytic activity of other endonucleases such asRNase H or restriction endonucleases to the same extent, preferably notat all. For example, the skilled person may set up the following sampleswith the same buffer and reaction conditions as well as substrate andendonuclease concentrations: (1) substrate such as panhandle RNA,endonucleolytically active PA polypeptide fragment or variant thereof,(2) substrate such as panhandle RNA, endonucleolytically active PApolypeptide fragment or variant thereof, test compound, (3) substratesuch as panhandle RNA, reference endonuclease such as RNAse H, (4)substrate such as panhandle RNA, reference nucleotide such as RNAse H,test compound. After incubation of the samples, the skilled person mayanalyze the substrate, for example, by gel electrophoresis. Testcompounds which result in cleaved substrate in sample (2) and intactsubstrate in sample (4) are preferred.

The term “in a high-throughput setting” refers to high-throughputscreening assays and techniques of various types which are used toscreen libraries of test compounds for their ability to inhibit theendonuclease activity of the polypeptide fragment of interest.Typically, the high-throughput assays are performed in a multi-wellformat and include cell-free as well as cell-based assays.

The term “antibody” refers to both monoclonal and polyclonal antibodies,i.e., any immunoglobulin protein or portion thereof which is capable ofrecognizing an antigen or hapten, i.e., the PA polypeptide fragmentpossessing endonucleolytic activity or a peptide thereof. In a preferredembodiment, the antibody is capable of binding to the enzymatically(endonucleolytically) active center within the PA polypeptide fragmentor variant thereof Antigen-binding portions of the antibody may beproduced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. In some embodiments, antigen-bindingportions include Fab, Fab', F(ab')₂, Fd, Fv, dAb, and complementaritydetermining region (CDR) fragments, single-chain antibodies (scFv),chimeric antibodies such as humanized antibodies, diabodies, andpolypeptides that contain at least a portion of an antibody that issufficient to confer specific antigen binding to the polypeptide.

The term “pharmaceutically acceptable salt” refers to a salt of acompound identifiable by the methods of the present invention or acompound of the present invention. Suitable pharmaceutically acceptablesalts include acid addition salts which may, for example, be formed bymixing a solution of compounds of the present invention with a solutionof a pharmaceutically acceptable acid such as hydrochloric acid,sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid,benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoricacid. Furthermore, where the compound carries an acidic moiety, suitablepharmaceutically acceptable salts thereof may include alkali metal salts(e.g., sodium or potassium salts); alkaline earth metal salts (e.g.,calcium or magnesium salts); and salts formed with suitable organicligands (e.g., ammonium, quaternary ammonium and amine cations formedusing counteranions such as halide, hydroxide, carboxylate, sulfate,phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrativeexamples of pharmaceutically acceptable salts include, but are notlimited to, acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, butyrate, calcium edetate, camphorate, camphorsulfonate,camsylate, carbonate, chloride, citrate, clavulanate,cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate,edetate, edisylate, estolate, esylate, ethanesulfonate, formate,fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like(see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19 (1977)).

The term “excipient” when used herein is intended to indicate allsubstances in a pharmaceutical formulation which are not activeingredients such as, e.g., carriers, binders, lubricants, thickeners,surface active agents, preservatives, emulsifiers, buffers, flavoringagents, or colorants.

The term “pharmaceutically acceptable carrier” includes, for example,magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

DETAILED DESCRIPTION

The present invention establishes for the first time a unique role forthe PA subunit of influenza virus polymerase and contradicts the widelyheld view that the endonuclease active site is located within the PB 1subunit. The present inventors surprisingly found that a smallindependently folded domain derived from the N-terminus of the PAsubunit exhibits the functional properties of the endonuclease reportedfor the trimeric complex, although this activity was thought to bedetectable only in the trimeric complex. Moreover, the inventors foundthat this PA polypeptide fragment can easily be produced by recombinantmeans and thus is suitable for in vitro studies on the endonucleolyticactivity and and its modulation as well as for crystallization to obtainstructural information in particular on the active site.

It is one aspect of the present invention to provide a polypeptidefragment comprising an amino-terminal fragment of the PA subunit of aviral RNA-dependent RNA polymerase possessing endonuclease activity,wherein said PA subunit is from a virus belonging to theOrthomyxoviridae family. Preferably, the polypeptide fragment is solublein an aqueous solution. The minimal length of the polypeptide fragmentof the present invention is determined by its ability to cleavepolynucleotide chains such as panhandle RNA or single stranded DNA,i.e., the minimal length of the polypeptide is determined by itsendonucleolytic activity. Preferably, the endonuclease activity is notdependent on the polynucleotide type, and thus, may be exerted on DNAand RNA, preferably on single stranded DNA and RNA. Preferably, theendonuclease activity is not dependent on specific recognition siteswithin the substrate polynucleotide.

In a preferred embodiment, the polypeptide fragment is suitable forcrystallization, i.e., preferably the polypeptide fragment iscrystallizable. Preferably, the crystals obtainable from the polypeptidefragment according to the invention are suitable for structuredetermination of the polypeptide fragment using X-ray crystallography.Preferably, said crystals are greater than 25 micron cubes andpreferably are radiation stable enough to permit more than 85%diffraction data completeness at resolution of preferably 3.5 Å orbetter to be collected upon exposure to monochromatic X-rays.

In one embodiment, the polypeptide fragment is crystallizable using (i)an aqueous protein solution, i.e., the crystallization solution, with aprotein concentration of 5 to 10 mg/ml, e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, or 10 mg/ml, preferably of 8 to 10 mg/ml in a buffer systemsuch as Tris-HC1 at concentrations ranging from 10 mM to 3 M, preferably10 mM to 2 M, more preferably 20 mM to 1 M, at pH 3 to pH 9, preferablypH 4 to pH 9, more preferably pH 7 to pH 9 and (ii) aprecipitant/reservoir solution comprising one or more compounds such assodium formate, ammonium sulphate, lithium sulphate, magnesium acetate,manganese acetate, or ethylene glycol. Optionally, the protein solutionmay contain one or more salts such as monovalent salts, e.g., NaCl, KCl,or LiCl, preferably NaCl, at concentrations ranging from 10 mM to 1 M,preferably 20 mM to 500 mM, more preferably 50 mM to 200 mM, and/ordivalent salts, e.g., MnCl₂, CaCl₂, MgCl₂, ZnCl₂, or CoCl₂, preferablyMnCl₂, at concentrations ranging from 0.1 to 50 mM, preferably 0.5 to 25mM, more preferably 1 to 10 mM. Preferably, the precipitant/reservoirsolution comprises Li₂SO₄ at concentrations ranging from 0.5 to 2 M,preferably 1 to 1.5 M, a buffer system such as MES at concentrationsranging from 20 mM to 1 M, preferably 50 mM to 500 mM, more preferably75 to 150 mM, at preferably pH 4 to 8, more preferably pH 5 to 7,magnesium acetate and/or manganese acetate at concentrations rangingfrom 1 to 100 mM, preferably from 5 to 20 mM, and/or ethylene glycol atconcentrations ranging from 1% to 20%, preferably 2% to 8%, morepreferably 2 to 4%. The PA polypeptide fragment or variant thereof ispreferably 85% to 100% pure, more preferably 90% to 100% pure, even morepreferably 95% to 100% pure in the crystallization solution. To producecrystals, the protein solution suitable for crystallization may be mixedwith an equal volume of the precipitant solution. In a preferredembodiment, the crystallization medium comprises 0.05 to 2 μl,preferably 0.8 to 1.2 μl, of protein solution suitable forcrystallization mixed with a similar, preferably equal volume ofprecipitant solution comprising 1.0 to 1.4 M Li₂SO₄, 80 to 120 mM MES pH5.5 to pH 6.5, 5 to 15 mM magnesium acetate and/or manganese acetate,and 2 to 4% ethylene glycol. In another embodiment, the precipitantsolution comprises, preferably essentially consists of or consists of1.2 M Li₂SO₄, 100 mM MES pH 6.0, 10 mM magnesium acetate and/or 10 mMmanganese acetate, preferably 10 mM magnesium acetate, and 3% ethyleneglycol, and the crystallization/protein solution comprises, preferablyessentially consists or consists of 5 to 10 mg/ml protein, 20 mM Tris pH8.0, 100 mM NaCl, and 2.5 mM MnCl₂.

Crystals can be grown by any method known to the person skilled in theart including, but not limited to, hanging and sitting drop techniques,sandwich-drop, dialysis, and microbatch or microtube batch devices. Itwould be readily apparent to one of skill in the art to vary thecrystallization conditions disclosed above to identify othercrystallization conditions that would produce crystals of PA polypeptidefragments of the inventions or variants thereof alone or in complex witha compound. Such variations include, but are not limited to, adjustingpH, protein concentration and/or crystallization temperature, changingthe identity or concentration of salt and/or precipitant used, using adifferent method for crystallization, or introducing additives such asdetergents (e.g., TWEEN 20 (monolaurate),

LDOA, Brij 30 (4 lauryl ether)), sugars (e.g., glucose, maltose),organic compounds (e.g., dioxane, dimethylformamide), lanthanide ions,or poly-ionic compounds that aid in crystallizations. High throughputcrystallization assays may also be used to assist in finding oroptimizing the crystallization condition.

Microseeding may be used to increase the size and quality of crystals.In brief, micro-crystals are crushed to yield a stock seed solution. Thestock seed solution is diluted in series. Using a needle, glass rod orstrand of hair, a small sample from each diluted solution is added to aset of equilibrated drops containing a protein concentration equal to orless than a concentration needed to create crystals without the presenceof seeds. The aim is to end up with a single seed crystal that will actto nucleate crystal growth in the drop.

The manner of obtaining the structure coordinates as shown in FIG. 18,interpretation of the coordinates and their utility in understanding theprotein structure, as described herein, are commonly understood by theskilled person and by reference to standard texts such as J. Drenth,“Principles of protein X-ray crystallography”, 2^(nd) Ed., SpringerAdvanced Texts in Chemistry, New York (1999); and G. E. Schulz and R. H.Schirmer, “Principles of Protein Structure”, Springer Verlag, New York(1985). For example, X-ray diffraction data is first acquired, oftenusing cryoprotected (e.g., with 20% to 30% glycerol) crystals frozen to100 K, e.g., using a beamline at a synchrotron facility or a rotatinganode as an X-ray source. Then, the phase problem is solved by agenerally known method, e.g., multiwavelength anomalous diffraction(MAD), multiple isomorphous replacement (MIR), single wavelengthanomalous diffraction (SAD), or molecular replacement (MR). Thesub-structure may be solved using SHELXD (Schneider and Sheldrick, 2002,Acta Crystallogr. D. Biol. Crystallogr. (Pt 10 Pt 2), 1772-1779), phasescalculated with SHARP (Vonrhein et al., 2006, Methods Mol. Biol.364:215-30), and improved with solvent flattening andnon-crystallographic symmetry averaging, e.g., with RESOLVE(Terwilliger, 2000, Acta Cryst. D. Biol. Crystallogr. 56:965-972). Modelautobuilding can be done, e.g., with ARP/wARP (Perrakis et al., 1999,Nat. Struct. Biol. 6:458-63) and refinement with, e.g., REFMAC(Murshudov, 1997, Acta Crystallogr. D. Biol. Crystallogr. 53: 240-255).The skilled person can use the structure coordinates (FIG. 18) as inputfor secondary analysis, including the determination of electrostaticsurface potential (see FIG. 13), which aids in the determination of sidegroups in test compounds, which are likely to interact with a surfacearea of the PA of a given electrostatic potential, preferably in theactive site. In order to use the structure coordinates generated for thePA polypeptide fragment it is necessary to convert the structurecoordinates into a three-dimensional shape. This is achieved through theuse of commercially available software that is capable of generatingthree-dimensional graphical representations of molecules or portionsthereof from a set of structure coordinates. An example for such acomputer program is MODELER (Sali and Blundell, 1993, J. Mol. Biol.234:779-815 as implemented in the Insight II Homology software package(Insight II (97.0),

Molecular Simulations Incorporated, San Diego, Calif.)). Such athree-dimensional graphical representations can be use with suitableprograms including (i) Gaussian 92, revision C (Frisch, Gaussian,Incorporated, Pittsburgh, Pa.), (ii) AMBER, version 4.0 (Kollman,University of California, San Francisco, Calif.), (iii) QUANTA/CHARMM(Molecular Simulations Incorporated, San Diego, Calif.), (iv) OPLS-AA(Jorgensen, 1998, Encyclopedia of Computational Chemistry, Schleyer,Ed., Wiley, N.Y., Vol. 3, pp. 1986-1989), and (v) Insight II/Discover(Biosysm Technologies Incorporated, San Diego, Calif.) to generategraphic representations of, e.g. electrostatic potential. Similarly, thestructural information can be combined with information on theconservation of residues as depicted in FIG. 11 at the various aminoacid positions (see FIG. 12) to highlight those residues at the surfaceof the PA and/or in the active site, which are particularly conservedbetween different virus isolates and, consequently, are likely to bealso present in mutants of thoses viruses or other isolates. Thissuitable in the the skilled person is able to derive information on therelevance of the residues Furthermore, the structure coordinates (FIG.18) of the Influenza A virus PA fragment PA-Nter provided by the presentinvention are useful for the structure determination of PA polypeptidesof other viruses from the Orthomyxoviridae family, or PA polypeptidevariants that have amino acid substitutions, deletions, and/orinsertions using the method of molecular replacement.

In a preferred embodiment of the polypeptide fragment according to theinvention, the

PA subunit is from Influenza A, B, or C virus or is a variant thereof,preferably from Influenza A virus or a variant thereof. Preferably, theamino terminal PA fragment comprised within the polypeptide fragmentaccording to the present invention corresponds to, preferablyessentially consists or consists of at least amino acids 1 to 196,preferably amino acids 1 to 209, preferably amino acids 1 to 213 of thePA subunit of the RNA-dependent RNA polymerase of Influenza A virus orvariants thereof, i.e., amino acid residues 1 to 196, 1 to 209, or 1 to213 of the amino acid sequence as set forth in SEQ ID NO: 2.

In a preferred embodiment, the polypeptide fragment according to thepresent invention is purified to an extent to be suitable forcrystallization, preferably it is 85% to 100%, more preferably 90% to100%, most preferably 95% to 100% pure.

In another embodiment, the polypeptide fragment according to theinvention is capable of binding to divalent cations. Preferably, thepolypeptide fragment according to the present invention is bound to oneor more divalent cation(s), preferably it is bound to two divalentcations. In this context, the divalent cation is preferably selectedform the group consisting of manganese, cobalt, calcium, magnesium, andzinc, and is more preferably manganese or cobalt, most preferablymanganese. Thus, in a preferred embodiment, the polypeptide of thepresent invention is present in complex with two manganese cations. In apreferred embodiment, the divalent cations are coordinated by aminoacids corresponding to amino acids Glu80 and Asp108 (first cation) andamino acids corresponding to amino acids His41, Asp108, and Glul 19(second cation) as set forth in SEQ ID NO: 2.

In a preferred embodiment of the polypeptide fragment according to thepresent invention, (i) the N-terminus is identical to or corresponds toamino acid position 15 or lower, e.g., at position 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and the C-terminus is identical to orcorresponds to an amino acid at a position selected from positions 186to 220, e.g., 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, or 220 of the amino acidsequence of the PA subunit according to SEQ ID NO: 2; preferably theN-terminus is identical to or corresponds to amino acid position 15 orlower, e.g., at position 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 and the C-terminus is identical to or corresponds to an amino acidat a position selected from 196 to 220 of the amino acid sequence of thePA subunit according to SEQ ID NO: 2; more preferably the N-terminus isidentical to or corresponds to amino acid position 15 or lower, e.g., atposition 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and theC-terminus is identical to or corresponds to an amino acid at a positionselected from 196 to 209 of the amino acid sequence of the PA subunitaccording to SEQ ID NO: 2, (ii) the N-terminus is identical to orcorresponds to amino acid position 15 or lower, e.g., amino acidposition 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and theC-terminus is identical to or corresponds to an amino acid at a positionselected from positions 185 to 217, e.g., 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217 ofthe amino acid sequence of the PA subunit according to SEQ ID NO: 4;preferably the N-terminus is identical to or corresponds to amino acidposition 15 or lower, e.g., amino acid position 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1, and the C-terminus is identical to orcorresponds to an amino acid at a position selected from positions 195to 217 of the amino acid sequence of the PA subunit according to SEQ IDNO: 4; more preferably the N-terminus is identical to or corresponds toamino acid position 15 or lower, e.g., amino acid position 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and the C-terminus isidentical to or corresponds to an amino acid at a position 195 to 206 ofthe amino acid sequence according to SEQ ID NO: 4, or (iii) theN-terminus is identical to or corresponds to amino acid position 15 orlower, e.g., amino acid position 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1, and the C-terminus is identical to or corresponds to anamino acid at a position selected from positions 168 to 200, e.g., aminoacid position 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, or 200 of the amino acid sequence ofthe PA subunit according to SEQ ID NO: 6; preferably the N-terminus isidentical to or corresponds to amino acid position 15 or lower, e.g.,amino acid position 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1, and the C-terminus is identical to or corresponds to an amino acid ata position selected from positions 178 to 200 of the amino acid sequenceaccording to SEQ ID NO: 6, and variants thereof, which retain theendonuclease activity; more preferably the N-terminus is identical to orcorresponds to amino acid position 15 or lower, e.g., amino acidposition 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and theC-terminus is identical to or corresponds to an amino acid at a positionselected from positions 178 to 189 of the amino acid sequence accordingto SEQ ID NO: 6; and in each case variants of the amino acid sequenceaccording to SEQ ID NO: 2, 4 or 6, which retain endonuclease activity.

In another embodiment said polypeptide fragment has or corresponds to anamino acid sequence selected from the group of amino acid sequencesconsisting of amino acids 5 to 196, 10 to 196, 15 to 196, 20 to 196, 5to 209, 10 to 209, 15 to 209, 20 to 209 of the amino acid sequence setforth in SEQ ID NO: 2 and variants thereof, which retain theendonucleolytic activity. In another embodiment said PA polypeptidefragment has or corresponds to amino acids selected from the group ofamino acid sequences consisting of amino acids 5 to 195, 10 to 195, 15to 195, 20 to 195, 5 to 206, 10 to 206, 15 to 206, 20 to 206 of theamino acid sequence set forth in SEQ ID NO: 4 and variants thereof,which retain the endonucleolytic activity. In another embodiment said PApolypeptide fragment has or corresponds to amino acids selected from thegroup of amino acid sequences consisting of amino acids 5 to 178, 10 to178, 15 to 178, 20 to 178, 5 to 189, 10 to 189, 15 to 189, 20 to 189 ofthe amino acid sequence set forth in SEQ ID NO: 6 and variants thereof,which retain the endonucleolytic activity. In preferred embodiments,said polypeptide fragments comprise amino acid substitutions,insertions, or deletions, preferably naturally occurring mutations asset forth above.

In another preferred embodiment, the polypeptide fragment according tothe present invention consists of amino acids 1 to 209 of the amino acidsequence set forth in SEQ ID NO: 2 and has the structure defined by thestructure coordinates as shown in FIG. 18.

In another embodiment, the polypeptide fragment according to the presentinvention has a crystalline form, preferably with space group P4₃2₁2,with unit cell dimensions of preferably a=b=6.71±0.2 nm, c=30.29 nm±0.4nm. In another embodiment, the crystals according to the invention arehexagonal plates with preferred unit cell dimensions of a=b=6.79 nm,c=49.4 nm, α=β=90°, and γ=120° having preferably a trigonal or hexagonalspace group. Preferably, the crystal of the polypeptide fragmentdiffracts X-rays to a resolution of 2.8 Å or higher, preferably 2.6 Å orhigher, more preferably 2.5 Å or higher, even more preferably 2.4 Å orhigher, most preferably 2.1 Å or higher.

It is another aspect of the present invention to provide an isolatedpolynucleotide coding for the above-mentioned PA polypeptide fragmentsand variants thereof. The molecular biology methods applied forobtaining such isolated nucleotide fragments are generally known to theperson skilled in the art (for standard molecular biology methods seeSambrook et al., Eds., “Molecular Cloning: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), whichis incorporated herein by reference). For example, RNA can be isolatedfrom Influenza virus infected cells and cDNA generated applying reversetranscription polymerase chain reaction (RT-PCR) using either randomprimers (e.g., random hexamers of decamers) or primers specific for thegeneration of the fragments of interest. The fragments of interest canthen be amplified by standard PCR using fragment specific primers.

In a preferred embodiment the isolated polynucleotide coding for thepreferred embodiments of the PA polypeptide fragments are derived fromSEQ ID NO: 1 (Influenza A), 3 (Influenza B), or 6 (Influenza C). In thiscontext, “derived” refers to the fact that SEQ ID NO: 1, 2, and 3 encodethe full-length PA polypeptides and, thus, polynucleotides coding forpreferred PA polypeptide fragments may comprise deletions at the 3′-and/or 5′-ends of the polynucleotide as required by the respectivelyencoded PA polypeptide fragment.

In one embodiment, the present invention relates to a recombinant vectorcomprising said isolated polynucleotide. The person skilled in the artis well aware of techniques used for the incorporation of polynucleotidesequences of interest into vectors (also see Sambrook et al., 1989,supra). Such vectors include any vectors known to the skilled personincluding plasmid vectors, cosmid vectors, phage vectors such as lambdaphage, viral vectors such as adenoviral or baculoviral vectors, orartificial chromosome vectors such as bacterial artificial chromosomes(BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes(PAC). Said vectors may be expression vectors suitable for prokaryoticor eukaryotic expression. Said plasmids may include an origin ofreplication (ori), a multiple cloning site, and regulatory sequencessuch as promoter (constitutive or inducible), transcription initiationsite, ribosomal binding site, transcription termination site,polyadenylation signal, and selection marker such as antibioticresistance or auxotrophic marker based on complementation of a mutationor deletion. In one embodiment the polynucleotide sequence of interestis operably linked to the regulatory sequences.

In another embodiment, said vector includes nucleotide sequences codingfor epitope-, peptide-, or protein-tags that facilitate purification ofpolypeptide fragments of interest. Such epitope-, peptide-, orprotein-tags include, but are not limited to, hemagglutinin- (HA-),FLAG-, myc-tag, poly-His-tag, glutathione-S-transferase- (GST-),maltose-binding-protein-(MBP-), NusA-, and thioredoxin-tag, orfluorescent protein-tags such as (enhanced) green fluorescent protein((E)GFP), (enhanced) yellow fluorescent protein ((E)YFP), redfluorescent protein (RFP) derived from Discosoma species (DsRed) ormonomeric (mRFP), cyan fluorescence protein (CFP), and the like. In apreferred embodiment, the epitope-, peptide-, or protein-tags can becleaved off the polypeptide fragment of interest, for example, using aprotease such as thrombin, Factor Xa, PreScission, TEV protease, and thelike. Preferably, the tag can be cleaved of with a TEV protease. Therecognition sites for such proteases are well known to the personskilled in the art. For example, the seven amino acid consensus sequenceof the TEV protease recognition site is Glu-X—X-Tyr-X-Gln-Gly/Ser,wherein X may be any amino acid and is in the context of the presentinvention preferably Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 21). Inanother embodiment, the vector includes functional sequences that leadto secretion of the polypeptide fragment of interest into the culturemedium of the recombinant host cells or into the periplasmic space ofbacteria. The signal sequence fragment usually encodes a signal peptidecomprised of hydrophobic amino acids which direct the secretion of theprotein from the cell. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria). Preferably there are processing sites, which can be cleavedeither in vivo or in vitro encoded between the signal peptide fragmentand the foreign gene.

In another aspect, the present invention provides a recombinant hostcell comprising said isolated polynucleotide or said recombinant vector.The recombinant host cells may be prokaryotic cells such as archea andbacterial cells or eukaryotic cells such as yeast, plant, insect, ormammalian cells. In a preferred embodiment the host cell is a bacterialcell such as an E. coli cell. The person skilled in the art is wellaware of methods for introducing said isolated polynucleotide or saidrecombinant vector into said host cell. For example, bacterial cells canbe readily transformed using, for example, chemical transformation,e.g., the calcium chloride method, or electroporation. Yeast cells maybe transformed, for example, using the lithium acetate transformationmethod or electroporation. Other eukaryotic cells can be transfected,for example, using commercially available liposome-based transfectionkits such as Lipofectamine™ (Invitrogen), commercially availablelipid-based transfection kits such as Fugene (Roche Diagnostics),polyethylene glycol-based transfection, calcium phosphate precipitation,gene gun (biolistic), electroporation, or viral infection. In apreferred embodiment of the invention, the recombinant host cellexpresses the polynucleotide fragment of interest. In an even morepreferred embodiment, said expression leads to soluble polypeptidefragments of the invention. These polypeptide fragments may be purifiedusing protein purification methods well known to the person skilled inthe art, optionally taking advantage of the above-mentioned epitope-,peptide-, or protein-tags.

In another aspect, the present invention relates to a method foridentifying compounds which modulate the endonuclease activity of the PAsubunit of a viral RNA-dependent RNA polymerase from theOrthomyxoviridae family or a variant thereof, comprising the steps of

(a) constructing a computer model of the active site defined by thestructure coordinates of the polypeptide fragment according to thepresent invention shown in FIG. 18;(b) selecting a potential activity modulating compound by a methodselected from the group consisting of:

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

(c) employing computational means to perform a fitting program operationbetween computer models of the said compound and the said active site inorder to provide an energy-minimized configuration of the said compoundin the active site; and(d) evaluating the results of said fitting operation to quantify theassociation between the said compound and the active site model, wherebyevaluating the ability of said compound to associate with the saidactive site.

Preferably, the modulating compound binds to the endonucleolyticallyactive site within the PA subunit or variant thereof. The modulatingcompound may increase or decrease, preferably decrease saidendonucleolytic activity.

In a preferred embodiment of this aspect of the present invention, thecompound that modulates the endonuclease activity of the PA subunit or avariant thereof decreases said activity, more preferably said compoundinhibits said activity. Preferably, the compound decreases theendonucleolytic activity of the PA subunit or a variant thereof by 50%,more preferably by 60%, even more preferably by 70%, even morepreferably by 80%, even more preferably by 90%, and most preferably by100% compared to the endonucleolytic activity of the PA subunit or avariant thereof without said compound but with otherwise the samereaction conditions, i.e., buffer conditions, reaction time andtemperature. It is particularly preferred that the compound specificallydecreases or inhibits the endonucleolytic activity of the PA subunit ora variant thereof but does not decrease or inhibit the endonucleolyticactivity of other endonucleases, in particular of mammalianendonucleases, to the same extent, preferably not at all.

For the first time, the present invention permits the use of moleculardesign techniques to identify, select, or design of compoundspotentially modulating the endonucleolytic activity of the PA subunit orvariants thereof, based on the structure coordinates of theendonucleolytically active site according to FIG. 18. Such a predictivemodel is valuable in light of the higher costs associated with thepreparation and testing of the many diverse compounds that may possiblymodulate the endonucleolytic activity. In order to use the structurecoordinates generated for the PA polypeptide fragment it is necessary toconvert the structure coordinates into a three-dimensional shape. Thisis achieved through the use of commercially available software that iscapable of generating three-dimensional graphical representations ofmolecules or portions thereof from a set of structure coordinates. Anexample for such a computer program is MODELER (Sali and Blundell, 1993,J. Mol. Biol. 234:779-815 as implemented in the Insight II Homologysoftware package (Insight II (97.0), Molecular Simulations Incorporated,San Diego, Calif.)).

One skilled in the art may use several methods to screen chemicalentities or fragments for their ability to modulate the endonucleolyticactivity of the PA subunit or PA polypeptide variants. This process maybegin by a visual inspection of, for example, a three-dimensionalcomputer model of the endonucleolytically active site of PA based on thestructural coordinates according to FIG. 18. Selected fragments orchemical compounds may then be positioned in a variety of orientationsor docked within the active site. Docking may be accomplished usingsoftware such as Cerius, Quanta, and Sybyl (Tripos Associates, St.Louis, MO), followed by energy minimization and molecular dynamics withstandard molecular dynamics force fields such as OPLS-AA, CHARMM, andAMBER. Additional specialized computer programs that may assist theperson skilled in the art in the process of selecting suitable compoundsor fragments include, for example, (i) AUTODOCK (Goodsell et al., 1990,Proteins: Struct., Funct., Genet. 8: 195-202; AUTODOCK is available fromThe Scripps Research Institute, La Jolla, Calif.) and (ii) DOCK (Kuntzet al., 1982, J. Mol. Biol.

161:269-288; DOCK is available from the University of California, SanFrancisco, Calif.).

Once suitable compounds or fragments have been selected, they can bedesigned or assembled into a single compound or complex. This manualmodel building is performed using software such as Quanta or Sybyl.Useful programs aiding the skilled person in connecting individualcompounds or fragments include, for example, (i) CAVEAT (Bartlett etal., 1989, in Molecular Recognition in Chemical and Biological Problems,Special Publication, Royal Chem. Soc. 78:182-196; Lauri and Bartlett,1994, J. Comp. Aid. Mol. Des. 8:51-66; CAVEAT is available from theUniversity of California, Berkley, CA), (ii) 3D Database systems such asISIS (MDL Information Systems, San Leandro, Calif.; reviewed in

Martin, 1992, J. Med. Chem. 35:2145-2154), and (iii) HOOK (Eisen et al.,1994, Proteins: Struct., Funct., Genet. 19:199-221; HOOK is availablefrom Molecular Simulations Incorporated, San Diego, Calif.).

Another approach enabled by this invention, is the computationalscreening of small molecule databases for compounds that can bind inwhole or part to the endonucleolytically active site of the PA subunitor active sites of PA polypeptide variants. In this screening, thequality of fit of such compounds to the active site may be judged eitherby shape complementarity or by estimated interaction energy (Meng etal., 1992, J. Comp. Chem. 13:505-524).

Alternatively, a potential modulator for the endonucleolytic activity ofthe PA subunit or polypeptide variant thereof, preferably an inhibitorof the endonucleolytic activity, may be designed de novo on the basis ofthe 3D structure of the PA polypeptide fragment according to FIG. 18.There are various de novo ligand design methods available to the personskilled in the art. Such methods include (i) LUDI (Bohm, 1992, J. Comp.Aid. Mol. Des. 6:61-78; LUDI is available from Molecular SimulationsIncorporated, San Diego, Calif.), (ii) LEGEND

(Nishibata and Itai, Tetrahedron 47:8985-8990; LEGEND is available fromMolecular Simulations Incorporated, San Diego, Calif.), (iii) LeapFrog(available from Tripos Associates, St. Louis, Mo.), (iv) SPROUT (Gilletet al., 1993, J. Comp. Aid. Mol. Des. 7:127-153; SPROUT is availablefrom the University of Leeds, UK), (v) GROUPBUILD (Rotstein and Murcko,1993, J. Med. Chem. 36:1700-1710), and (vi) GROW (Moon and Howe, 1991,

Proteins 11:314-328).

In addition, several molecular modeling techniques (hereby incorporatedby reference) that may support the person skilled in the art in de novodesign and modeling of potential modulators and/or inhibitors of theendonucleolytically active site, preferably binding partners of theendonucleolytically active site, have been described and include, forexample, Cohen et al., 1990, J. Med. Chem. 33:883-894; Navia and Murcko,1992, Curr. Opin. Struct. Biol. 2:202-210; Balbes et al., 1994, Reviewsin Computational Chemistry, Vol. 5, Lipkowitz and Boyd, Eds., VCH, NewYork, pp. 37-380; Guida, 1994, Curr. Opin. Struct. Biol. 4:777-781.

A molecule designed or selected as binding to the endonucleolyticallyactive site of the PA subunit or variants thereof may be furthercomputationally optimized so that in its bound state it preferably lacksrepulsive electrostatic interaction with the target region. Suchnon-complementary (e.g., electrostatic) interactions include repulsivecharge-charge, dipole-dipole and charge-dipole interactions.Specifically, the sum of all electrostatic interactions between thebinding compound and the binding pocket in a bound state, preferablymake a neutral or favorable contribution to the enthalpy of binding.Specific computer programs that can evaluate a compound deformationenergy and electrostatic interaction are available in the art. Examplesof suitable programs include (i) Gaussian 92, revision C (Frisch,Gaussian, Incorporated, Pittsburgh, PA), (ii) AMBER, version 4.0(Kollman, University of California,

San Francisco, Calif.), (iii) QUANTA/CHARMM (Molecular SimulationsIncorporated, San Diego, Calif.), (iv) OPLS-AA (Jorgensen, 1998,Encyclopedia of Computational Chemistry, Schleyer, Ed., Wiley, New York,Vol. 3, pp. 1986-1989), and (v) Insight IUDiscover (Biosysm TechnologiesIncorporated, San Diego, Calif.). These programs may be implemented, forinstance, using a Silicon Graphics workstation, IRIS 4D/35 or IBMRISC/6000 workstation model 550. Other hardware systems and softwarepackages are known to those skilled in the art.

Once a molecule of interest has been selected or designed, as describedabove, substitutions may then be made in some of its atoms or sidegroups in order to improve or modify its binding properties. Generally,initial substitutions are conservative, i.e., the replacement group willapproximate the same size, shape, hydrophobicity and charge as theoriginal group. It should, of course, be understood that componentsknown in the art to alter conformation should be avoided. Suchsubstituted chemical compounds may then be analyzed for efficiency offit to the endonucleolytically active site of the PA subunit or variantthereof by the same computer methods described in detail above.

In one embodiment of the above-described method of the invention, theendonucleolytically active site of the PA subunit or variant thereofcomprises amino acids corresponding to amino acids Asp108, Ile120, andLys134 of the PA subunit according to SEQ ID NO: 2. In anotherembodiment, said active site comprises amino acids corresponding toamino acids Asp108, Ile120, Lys134, and His41 according to SEQ ID NO: 2.In another embodiment, said active site comprises amino acidscorresponding to amino acids Asp108, Ile120, Lys134, and Glu80 accordingto SEQ ID NO: 2. In another embodiment, said active site comprises aminoacids corresponding to amino acids Asp108, Ile120, Lys134, and Glu119according to SEQ ID NO: 2. In another embodiment, said active sitecomprises amino acids corresponding to amino acids Asp108, Ile120,Lys134, His41, Glu80, and Glu119 according to SEQ ID NO: 2. In yetanother embodiment, said active site comprises amino acids correspondingto amino acids Asp108, Ile120, Lys134, His41, Glu80, Glu119, and Tyr24according to SEQ ID NO: 2. In yet another embodiment, said active sitecomprises amino acids corresponding to amino acids Asp108, Ile120,Lys134, His41, Glu80, Glu119, and Arg84 according to SEQ ID NO: 2. Inyet another embodiment, said active site comprises amino acidscorresponding to amino acids Asp108, Ile120, Lys134, His41, Glu80,Glu119, and Leu106 according to SEQ ID NO: 2. In yet another embodiment,said active site comprises amino acids corresponding to amino acidsAsp108, Ile120, Lys134, His41, Glu80, Glu119, and Tyr130 according toSEQ ID NO: 2. In yet another embodiment, said active site comprisesamino acids corresponding to amino acids Asp108, Ile120, Lys134, His41,Glu80, Glu119, and Glu133 according to SEQ ID NO: 2. In yet anotherembodiment, said active site comprises amino acids corresponding toamino acids Asp108, Ile120, Lys134, His41, Glu80, Glu119, and Lys137according to SEQ ID NO: 2. In another embodiment, said active sitecomprises amino acids corresponding to amino acids Asp108, Ile 120,Lys134, His41, Glu80, Glu119, Tyr24, Arg84, and Leu106 according to SEQID NO: 2. In another embodiment, said active site comprises amino acidscorresponding to amino acids Asp108, Ile 120, Lys134, His41, Glu80,Glu119, Tyr130, Glu133, and Lys137 according to SEQ ID NO: 2. In anotherembodiment, said active site comprises amino acids corresponding toamino acids Asp108, Ile 120, Lys134, His41, Glu80, Glu119, Tyr24, Arg84,Leu106, Tyr130, Glu133, and Lys137 according to SEQ ID NO: 2.

In a further aspect of the above-described method of the invention, theendonucleolytically active site of the PA subunit or a variant thereofis defined by the structure coordinates of the PA SEQ ID NO: 2 aminoacids Asp108, Ile120, and Lys134 according to FIG. 18. In anotherembodiment, said active site is defined by the structure coordinates ofPA SEQ ID NO: 2 amino acids Asp108, Ile120, Lys134, and His41 accordingto FIG. 18. In another embodiment, said active site is defined by thestructure coordinates of PA SEQ ID NO: 2 amino acids Asp108, Ile120,Lys134, and Glu80 according to FIG. 18. In another embodiment, saidactive site is defined by the structure coordinates of PA SEQ ID NO: 2amino acids Asp108, Ile120, Lys134, and Glu119 according to FIG. 18. Inanother embodiment, said active site is defined by the structurecoordinates of PA SEQ ID NO: 2 amino acids Asp108, Ile120, Lys134,His41, Glu80, and Glu119 according to FIG. 18. In another embodiment,said active site is defined by the structure coordinates of PA SEQ IDNO: 2 amino acids Asp108, Ile120, Lys134, His41, Glu80, Glu119, andTyr24 according to FIG. 18. In yet another embodiment, said active siteis defined by the structure coordinates of PA SEQ ID NO: 2 amino acidsAsp108, Ile120, Lys134, His41, Glu80, Glul 19, and Arg84 according toFIG. 18. In another embodiment, said active site is defined by thestructure coordinates of PA SEQ ID NO: 2 amino acids Asp108, Ile120,Lys134, His41, Glu80, Glu119, and Leu106 according to FIG. 18. Inanother embodiment, said active site is defined by the structurecoordinates of PA SEQ ID NO: 2 amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Tyr130 according to FIG. 18. In anotherembodiment, said active site is defined by the structure coordinates ofPA SEQ ID NO: 2 amino acids Asp108, Ile120, Lys134, His41, Glu80,Glu119, and Glu133 according to FIG. 18. In another embodiment, saidactive site is defined by the structure coordinates of PA SEQ ID

NO: 2 amino acids Asp108, Ile120, Lys134, His41, Glu80, Glu119, andLys137 according to FIG. 18. In another embodiment, said active site isdefined by the structure coordinates of PA SEQ ID NO: 2 amino acidsAsp108, Ile120, Lys134, His41, Glu80, Glu119, Tyr24, Arg84, and Leu106according to FIG. 18. In another embodiment, said active site is definedby the structure coordinates of PA SEQ ID NO: 2 amino acids Asp108,Ile120, Lys134,

His41, Glu80, Glu119, Tyr130, Glu133, and Lys137 according to FIG. 18.In another embodiment, said active site is defined by the structurecoordinates of PA SEQ ID NO: 2 amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, Tyr24, Arg84, Leu106, Tyr130, Glu133, and Lys137according to FIG. 18.

In one aspect, the present invention provides a method for computationalscreening according to the above-described method for compounds able tomodulate and/or associate with an endonucleolytically active site thatis a variant to the endonucleolytically active site of the PA subunitaccording to FIG. 18. In one embodiment, said variant of said activesite has a root mean square deviation from the backbone atoms of aminoacids Asp108, Ile120, and Lys134, of amino acids Asp108, Ile120, Lys134,and His41, of amino acids Asp108, Ile120, Lys134, and Glu80, of aminoacids Asp108, Ile120, Lys134, and Glu119, of amino acids Asp108, Ile120,Lys134, His41, Glu80, and Glu119, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Tyr24, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Arg84, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Leu106, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Tyr130, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Glu133, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, and Lys137, of amino acids Asp108, Ile120, Lys134,His41, Glu80, Glu119, Tyr24, Arg84, and Leu106, of amino acids Asp108,Ile120, Lys134, His41, Glu80, Glu119, Tyr130, Glu133, and Lys137, ofamino acids Asp108, Ile120,

Lys134, His41, Glu80, Glu119, Tyr24, Arg84, Leu106, Tyr130, Glu133, andLys137 according to FIG. 18 of not more than 3 Å. In another embodiment,the said root mean square deviation is not more than 2.5 Å. In anotherembodiment, the said root mean square deviation is not more than 2 Å. Inanother embodiment, the said root mean square deviation is not more than1.5 Å. In another embodiment, the said root mean square deviation is notmore than 1 Å. In another embodiment, the said root mean squaredeviation is not more than 0.5 Å.

If computer modeling according to the methods described hereinaboveindicates binding of a compound to the active site of the PA subunit ora variant thereof, said compound may be synthesized and optionally saidcompound or a pharmaceutically acceptable salt thereof may be formulatedwith one or more pharmaceutically acceptable excipient(s) and/orcarrier(s). Thus, the above-described method may comprise the furtherstep of (e) synthesizing said compound and optionally formulating saidcompound or a pharmaceutically acceptable salt thereof with one or morepharmaceutically acceptable excipient(s) and/or carrier(s). Optionally,the ability of said compound or of a pharmaceutically acceptable saltthereof or of a formulation thereof to modulate, preferably decrease,preferably inhibit the endonucleolytic activity of the PA subunit orvariant thereof may be tested in vitro or in vivo comprising the furtherstep of (f) contacting said compound with the PA polypeptide fragment orvariant thereof or the recombinant host cell of the invention and todetermine the ability of said compound to (i) bind to the active siteand/or (ii) to modulate, decrease, or inhibit the endonucleolyticactivity of the PA subunit polypeptide fragment or variant thereof Thequality of fit of such compounds to the active site may be judged eitherby shape complementarity or by estimated interaction energy (Meng etal., 1992, J. Comp. Chem. 13:505-524). Methods for synthesizing saidcompounds are well known to the person skilled in the art or suchcompounds may be commercially available.

It is another aspect of the invention to provide a compound identifiableby the above-described method, wherein said compound is able to modulatethe endonuclease activity of the PA subunit or variant thereof Inanother aspect, the present invention refers to a compound identifiableby the above-described method, wherein said compound is able todecrease, preferably inhibit the endonuclease activity of the PA subunitor variant thereof, e.g., the PA subunit polypeptide or variant thereofaccording to the present invention.

Compounds of the present invention can be any agents including, but notrestricted to, peptides, peptoids, polypeptides, proteins (includingantibodies), lipids, metals, nucleotides, nucleosides, nucleic acids,small organic or inorganic molecules, chemical compounds, elements,saccharides, isotopes, carbohydrates, imaging agents, lipoproteins,glycoproteins, enzymes, analytical probes, polyamines, and combinationsand derivatives thereof The term “small molecules” refers to moleculesthat have a molecular weight between 50 and about 2,500 Daltons,preferably in the range of 200-800 Daltons. In addition, a test compoundaccording to the present invention may optionally comprise a detectablelabel. Such labels include, but are not limited to, enzymatic labels,radioisotope or radioactive compounds or elements, fluorescent compoundsor metals, chemiluminescent compounds and bioluminescent compounds. In apreferred embodiment of the compound according to the present invention,the compound is not a 4-substituted 2-dioxobutanoic acid, a4-substituted 4-dioxobutanoic acid, a 4-substituted 2,4-dioxobutanoicacid, a pyrazine-2,6-dione or a substituted pyrazine-2,6-dione such asflutimide, an N-hydroxamic acid, or an N-hydroxymide. In particular, thecompound according to the present invention is not a compound accordingto Formula I:

In a further aspect, the present invention provides a method foridentifying compounds which bind to the endonucleolytically active site,preferably modulate, more preferably decrease, most preferably inhibitthe endonuclease activity of the PA subunit or polypeptide variantsthereof, comprising the steps of (i) contacting the PA polypeptidefragment according to the present invention or a recombinant host cellaccording to the present invention with a test compound and (ii)analyzing the ability of said test compound to bind to theendonucleolytically active site, to modulate, to decrease, or to inhibitthe endonuclease activity of said PA subunit polypeptide fragment.

In one embodiment, the interaction between the PA polypeptide fragmentor variant thereof and a test compound may be analyzed in form of a pulldown assay. For example, the

PA polypeptide fragment may be purified and may be immobilized on beads.In one embodiment, the PA polypeptide fragment immobilized on beads maybe contacted, for example, with (i) another purified protein,polypeptide fragment, or peptide, (ii) a mixture of proteins,polypeptide fragments, or peptides, or (iii) a cell or tissue extract,and binding of proteins, polypeptide fragments, or peptides may beverified by polyacrylamide gel electrophoresis in combination withcoomassie staining or Western blotting. Unknown binding partners may beidentified by mass spectrometric analysis.

In another embodiment, the interaction between the PA polypeptidefragment or variant thereof and a test compound may be analyzed in formof an enzyme-linked immunosorbent assay (ELISA)-based experiment. In oneembodiment, the PA polypeptide fragment or variant thereof according tothe invention may be immobilized on the surface of an ELISA plate andcontacted with the test compound. Binding of the test compound may beverified, for example, for proteins, polypeptides, peptides, andepitope-tagged compounds by antibodies specific for the test compound orthe epitope-tag. These antibodies might be directly coupled to an enzymeor detected with a secondary antibody coupled to said enzyme that—incombination with the appropriate substrates—carries out chemiluminescentreactions (e.g., horseradish peroxidase) or colorimetric reactions(e.g., alkaline phosphatase). In another embodiment, binding ofcompounds that cannot be detected by antibodies might be verified bylabels directly coupled to the test compounds. Such labels may includeenzymatic labels, radioisotope or radioactive compounds or elements,fluorescent compounds or metals, chemiluminescent compounds andbioluminescent compounds. In another embodiment, the test compoundsmight be immobilized on the ELISA plate and contacted with the PApolypeptide fragment or variants thereof according to the invention.Binding of said polypeptide may be verified by a PA polypeptide fragmentspecific antibody and chemiluminescence or colorimetric reactions asdescribed above.

In a further embodiment, purified PA polypeptide fragments may beincubated with a peptide array and binding of the PA polypeptidefragments to specific peptide spots corresponding to a specific peptidesequence may be analyzed, for example, by PA polypeptide specificantibodies, antibodies that are directed against an epitope-tag fused tothe PA polypeptide fragment, or by a fluorescence signal emitted by afluorescent tag coupled to the PA polypeptide fragment.

In another embodiment, the recombinant host cell according to thepresent invention is contacted with a test compound. This may beachieved by co-expression of test proteins or polypeptides andverification of interaction, for example, by fluorescence resonanceenergy transfer (FRET) or co-immunoprecipitation. In another embodiment,directly labeled test compounds may be added to the medium of therecombinant host cells. The potential of the test compound to penetratemembranes and bind to the PA polypeptide fragment may be, for example,verified by immunoprecipitation of said polypeptide and verification ofthe presence of the label.

In another embodiment, the ability of the test compound to modulate,preferably decrease, more preferably inhibit the endonucleolyticactivity of the PA subunit polypeptide fragment or variant thereof isassessed. For example, the purified PA subunit polypeptide fragment anda substrate thereof such as panhandle RNA or single stranded DNA arecontacted in presence or absence of varying amounts of the test compoundand incubated for a certain period of time, for example, for 5, 10, 15,20, 30, 40, 60, or 90 minutes. The reaction conditions are chosen suchthat the PA subunit polypeptide is endonucleolytically active withoutthe test compound. The substrate is then analyzed fordegradation/endonucleolytic cleavage, for example, by gelelectrophoresis. Alternatively, such a test may comprise a labeledsubstrate molecule which provides a signal when the substrate moleculeis endonucleolytically cleaved but does not provide a signal if it isintact. For example, the substrate polynucleotide chain may be labeledwith fluorescent reporter molecule and a fluorescence quencher such thatthe fluorescent reporter is quenched as long as the substratepolynucleotide chain is intact. In case the substrate polynucleotidechain is cleaved, the fluorescent reporter and the quencher areseparated, thus, the fluorescent reporter emits a signal which may bedetected, for example, by an ELISA reader. This experimental setting maybe applied in a multi-well plate format and is suitable for highthroughput screening of compounds regarding their ability to modulate,decrease, or inhibit the endonuclease activity of the PA subunitpolypeptide fragment or variants thereof.

In a preferred embodiment, the above-described method for identifyingcompounds which associate with the endonucleolytically active site,modulate, decrease, or inhibit the endonucleolytic activity of the PAsubunit polypeptide fragment or variant thereof is performed in ahigh-throughput setting. In a preferred embodiment, said method iscarried out in a multi-well microtiter plate as described above using PApolypeptide fragments or variants thereof according to the presentinvention and labeled test compounds.

In a preferred embodiment, the test compounds are derived from librariesof synthetic or natural compounds. For instance, synthetic compoundlibraries are commercially available from Maybridge Chemical Co.(Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, Calif.),or Aldrich (Milwaukee, Wis.). A natural compound library is, forexample, available from TimTec LLC (Newark, Del.). Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts can be used. Additionally, test compounds can besynthetically produced using combinatorial chemistry either asindividual compounds or as mixtures.

In another embodiment, the inhibitory effect of the identified compoundon the Influenza virus life cycle may be tested in an in vivo setting. Acell line that is susceptible for Influenza virus infection such as 293Thuman embryonic kidney cells, Madin-Darby canine kidney cells, orchicken embryo fibroblasts may be infected with Influenza virus inpresence or absence of the identified compound. In a preferredembodiment, the identified compound may be added to the culture mediumof the cells in various concentrations. Viral plaque formation may beused as read out for the infectious capacity of the Influenza virus andmay be compared between cells that have been treated with the identifiedcompound and cells that have not been treated.

In a further embodiment of the invention, the test compound applied inany of the above described methods is a small molecule. In a preferredembodiment, said small molecule is derived from a library, e.g., a smallmolecule inhibitor library. In another embodiment, said test compound isa peptide or protein. In a preferred embodiment, said peptide or proteinis derived from a peptide or protein library.

In another embodiment of the above-described methods for computationalas well as in vitro identification of compounds that associate with theendonucleolytically active site, modulate, decrease, or inhibit theendonucleolytic activity of the PA subunit polypeptide fragment orvariant thereof according to the present invention, said methods furthercomprise the step of formulating the identifiable compound or apharmaceutically acceptable salt thereof with one or morepharmaceutically acceptable excipient(s) and/or carrier(s). In anotheraspect the present invention provides a pharmaceutical compositionproducible according to the afore-mentioned method. A compound accordingto the present invention can be administered alone but, in humantherapy, will generally be administered in admixture with a suitablepharmaceutical excipient, diluent, or carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice (see hereinafter).

In the aspect of computational modeling or screening of a bindingpartner for the endonucleolytically active site, a modulator, and/orinhibitor of the endonucleolytic activity of the PA subunit polypeptidefragment or variant thereof according to the present invention, it maybe possible to introduce into the molecule of interest, chemicalmoieties that may be beneficial for a molecule that is to beadministered as a pharmaceutical. For example, it may be possible tointroduce into or omit from the molecule of interest, chemical moietiesthat may not directly affect binding of the molecule to the target areabut which contribute, for example, to the overall solubility of themolecule in a pharmaceutically acceptable carrier, the bioavailabilityof the molecule and/or the toxicity of the molecule. Considerations andmethods for optimizing the pharmacology of the molecules of interest canbe found, for example, in “Goodman and Gilman's The PharmacologicalBasis of Therapeutics”, 8^(th) Edition, Goodman, Gilman, Rall, Nies, &Taylor, Eds., Pergamon Press (1985); Jorgensen & Duffy, 2000, Bioorg.Med. Chem. Lett. 10:1155-1158. Furthermore, the computer program “QikProp” can be used to provide rapid predictions for physicallysignificant descriptions and pharmaceutically-relevant properties of anorganic molecule of interest. A ‘Rule of Five’ probability scheme can beused to estimate oral absorption of the newly synthesized compounds(Lipinski et al., 1997, Adv. Drug Deliv. Rev. 23:3-25). Programssuitable for pharmacophore selection and design include (i) DISCO (AbbotLaboratories, Abbot Park, IL), (ii) Catalyst (Bio-CAD Corp., MountainView, Calif.), and (iii) Chem DBS-3D (Chemical

Design Ltd., Oxford, UK).

The pharmaceutical composition contemplated by the present invention maybe formulated in various ways well known to one of skill in the art. Forexample, the pharmaceutical composition of the present invention may bein solid form such as in the form of tablets, pills, capsules (includingsoft gel capsules), cachets, lozenges, ovules, powder, granules, orsuppositories, or in liquid form such as in the form of elixirs,solutions, emulsions, or suspensions.

Solid administration forms may contain excipients such asmicrocrystalline cellulose, lactose, sodium citrate, calcium carbonate,dibasic calcium phosphate, glycine, and starch (preferably corn, potato,or tapioca starch), disintegrants such as sodium starch glycolate,croscarmellose sodium, and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose(HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia.Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate, and talc may be included. Solid compositions ofa similar type may also be employed as fillers in gelatin capsules.Preferred excipients in this regard include lactose, starch, acellulose, milk sugar, or high molecular weight polyethylene glycols.

For aqueous suspensions, solutions, elixirs, and emulsions suitable fororal administration the compound may be combined with various sweeteningor flavoring agents, coloring matter or dyes, with emulsifying and/orsuspending agents and with diluents such as water, ethanol, propyleneglycol, and glycerin, and combinations thereof.

The pharmaceutical composition of the present invention may containrelease rate modifiers including, for example, hydroxypropylmethylcellulose, methyl cellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, Xanthan gum,

Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil,carnauba wax, paraffin wax, cellulose acetate phthalate,hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer, andmixtures thereof.

The pharmaceutical composition of the present invention may be in theform of fast dispersing or dissolving dosage formulations (FDDFs) andmay contain the following ingredients: aspartame, acesulfame potassium,citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethylacrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose,magnesium stearate, mannitol, methyl methacrylate, mint flavoring,polyethylene glycol, fumed silica, silicon dioxide, sodium starchglycolate, sodium stearyl fumarate, sorbitol, xylitol.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

The pharmaceutical composition of the present invention suitable forparenteral administration is best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary.

The pharmaceutical composition suitable for intranasal administrationand administration by inhalation is best delivered in the form of a drypowder inhaler or an aerosol spray from a pressurized container, pump,spray or nebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A.TM.) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA.TM.), carbon dioxide, oranother suitable gas. The pressurized container, pump, spray ornebulizer may contain a solution or suspension of the active compound,e.g., using a mixture of ethanol and the propellant as the solvent,which may additionally contain a lubricant, e.g., sorbitan trioleate.

It is another aspect of the invention to provide a compound identifiableby the above-described method, wherein the compound is able to modulatethe endonuclease activity of the PA subunit or variant thereof. Inanother aspect, the present invention refers to a compound identifiableby the above-described method, wherein the compound is able to decrease,preferably inhibit the endonuclease activity of the PA subunit orvariant thereof, e.g., the PA subunit polypeptide or variant thereofaccording to the present invention. Compounds of the present inventioncan be any agents as described above for the in silico screeningmethods. In a preferred embodiment of the compound according to thepresent invention, the compound is not a 4-substituted 2-dioxobutanoicacid, a 4-substituted 4-dioxobutanoic acid, a 4-substituted2,4-dioxobutanoic acid, a pyrazine-2,6-dione or a substitutedpyrazine-2,6-dione such as flutimide, an N-hydroxamic acid, or anN-hydroxymide. In particular, the compound according to the presentinvention is not a compound according to Formula I:

In another aspect, the present invention provides an antibody directedagainst the endonuclease domain of the PA subunit. In a preferredembodiment, said antibody recognizes the endonuclease domain byrecognition of a polypeptide fragment selected from the group ofpolypeptides defined by SEQ ID NO: 9 to 17, i.e., amino acids 20 to 30(SEQ ID NO: 9), 35 to 45 (SEQ ID NO: 10), 75 to 85 (SEQ ID NO: 11), 80to 90 (SEQ ID NO: 12), 100 to 110 (SEQ ID NO: 13), 107 to 112 (SEQ IDNO: 20), 115 to 125 (SEQ ID NO: 14), 125 to 135

(SEQ ID NO: 15), 130 to 140 (SEQ ID NO: 16), and 135 to 145 (SEQ ID NO:17) of the amino acid sequence as set forth in SEQ ID NO: 2. Preferablysaid antibody recognizes the amino sequence PDLYDYK (SEQ ID NO: 20). Inparticular, said antibody specifically binds to an epitope comprisingone or more of above indicated amino acids, which define the activesite. In this context, the term epitope has its art recognized meaningand preferably refers to stretches of 4 to 20 amino acids, preferably 5to 18, 5 to 15, or 7 to 14 amino acids. Accordingly, preferred epitopeshave a length of 4 to 20, 5 to 18, preferably 5 to 15, or 7 to 14 aminoacids and comprise one or more of Asp108, Ile120, Lys134, His41, Glu80,Glu119, Tyr24, Arg84, Leu106, Tyr130, Glu133, and/or Lys137 of SEQ IDNO: 2 or one or more corresponding amino acid(s).

The antibody of the present invention may be a monoclonal or polyclonalantibody or portions thereof. Antigen-binding portions may be producedby recombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some embodiments, antigen-binding portions includeFab, Fab', F(ab')₂, Fd, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodiessuch as humanized antibodies, diabodies, and polypeptides that containat least a portion of an antibody that is sufficient to confer specificantigen binding to the polypeptide. The antibody of the presentinvention is generated according to standard protocols. For example, apolyclonal antibody may be generated by immunizing an animal such asmouse, rat, rabbit, goat, sheep, pig, cattle, or horse with the antigenof interest optionally in combination with an adjuvant such as Freund'scomplete or incomplete adjuvant, RIBI (muramyl dipeptides), or ISCOM(immunostimulating complexes) according to standard methods well knownto the person skilled in the art. The polyclonal antiserum directedagainst the endonuclease domain of PA or fragments thereof is obtainedfrom the animal by bleeding or sacrificing the immunized animal. Theserum (i) may be used as it is obtained from the animal, (ii) animmunoglobulin fraction may be obtained from the serum, or (iii) theantibodies specific for the endonuclease domain of PA or fragmentsthereof may be purified from the serum. Monoclonal antibodies may begenerated by methods well known to the person skilled in the art. Inbrief, the animal is sacrificed after immunization and lymph node and/orsplenic B cells are immortalized by any means known in the art. Methodsof immortalizing cells include, but are not limited to, transfectingthem with oncogenes, infecting them with an oncogenic virus andcultivating them under conditions that select for immortalized cells,subjecting them to carcinogenic or mutating compounds, fusing them withan immortalized cell, e.g., a myeloma cell, and inactivating a tumorsuppressor gene. Immortalized cells are screened using the PAendonuclease domain or a fragment thereof Cells that produce antibodiesdirected against the

PA endonuclease domain or a fragment thereof, e.g., hybridomas, areselected, cloned, and further screened for desirable characteristicsincluding robust growth, high antibody production, and desirableantibody characteristics. Hybridomas can be expanded (i) in vivo insyngeneic animals, (ii) in animals that lack an immune system, e.g.,nude mice, or (iii) in cell culture in vitro. Methods of selecting,cloning, and expanding hybridomas are well known to those of ordinaryskill in the art. The skilled person may refer to standard texts such as“Antibodies: A Laboratory Manual”, Harlow and Lane, Eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which isincorporated herein by reference, for support regarding generation ofantibodies.

In another aspect, the present invention relates to the use of acompound identifiable by the above-described methods that is able tobind to the endonucleolytically active site of the PA subunitpolypeptide fragment or variant thereof, and/or is able to modulate,preferably decrease, more preferably inhibit the endonucleolyticactivity of the PA subunit polypeptide fragment or variant thereof, thepharmaceutical composition described above, or the antibody of thepresent invention for the manufacture of a medicament for treating,ameliorating, or preventing disease conditions caused by viralinfections with negative-sense single stranded RNA viruses of the familyof Orthomyxoviridae. In a preferred embodiment, said disease conditionsare caused by viral infections with Influenza A virus, Influenza Bvirus, Influenza C virus, Isavirus, or Thogotovirus. In an even morepreferred embodiment, said disease condition is caused by an infectionwith a virus species selected from the group consisting of Influenza Avirus, Influenza B virus, Influenza C virus, most preferably Influenza Avirus.

For treating, ameliorating, or preventing said disease conditions themedicament of the present invention can be administered to an animalpatient, preferably a mammalian patient, preferably a human patient,orally, buccally, sublingually, intranasally, via pulmonary routes suchas by inhalation, via rectal routes, or parenterally, for example,intracavernosally, intravenously, intra-arterially, intraperitoneally,intrathecally, intraventricularly, intra-urethrally intrasternally,intracranially, intramuscularly, or subcutaneously, they may beadministered by infusion or needleless injection techniques.

The pharmaceutical compositions of the present invention may beformulated in various ways well known to one of skill in the art and asdescribed above.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation administeredin the use of the present invention may be varied or adjusted from about1 mg to about 1000 mg per m², preferably about 5 mg to about 150 mg/m²according to the particular application and the potency of the activecomponent.

The compounds employed in the medical use of the invention areadministered at an initial dosage of about 0.05 mg/kg to about 20 mg/kgdaily. A daily dose range of about 0.05 mg/kg to about 2 mg/kg ispreferred, with a daily dose range of about 0.05 mg/kg to about 1 mg/kgbeing most preferred. The dosages, however, may be varied depending uponthe requirements of the patient, the severity of the condition beingtreated, and the compound being employed. Determination of the properdosage for a particular situation is within the skill of thepractitioner. Generally, treatment is initiated with smaller dosages,which are less than the optimum dose of the compound. Thereafter, thedosage is increased by small increments until the optimum effect undercircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day, if desired.

EXAMPLES

The Examples are designed in order to further illustrate the presentinvention and serve a better understanding. They are not to be construedas limiting the scope of the invention in any way.

Summary of the Examples

PA-Nter, residues 1-209 of the amino acid sequence set forth in SEQ IDNO: 2 (A/Victoria/3/1975 (H3N2)) was expressed in E. coli and purifiedby affinity and gel filtration chromatography. The influence of metalions on thermal stability was tested by thermofluor assays (Ericsson etal., 2006, Anal. Biochem. 357:289-298). The endonuclease activity wastested by incubation at 37° C. of 13 μM PA-Nter with 10 μM of variousRNA substrates: Alu-RNA; 110 nucleotides of the Alu-domain of P.horikoshii, SRP RNA, C. albicans tRNA Asn, U-rich RNA(5′-GGCCAUCCUGU₇CCCU₁₁CU₁₉-3′; SEQ ID NO: 18, Saito et al., 2008, Nature454:523-527), panhandle RNA (ph-RNA) of 81 nucleotides (Baudin et al.,1994,

EMBO J. 13:3158-3165), short ph-RNA of 36 nucleotides comprising justthe conserved 3′-and 5′-ends with a short linker, and circular singlestranded DNA (M13mp18) (Fermentas). Crystals diffracting to 2 Aresolution were obtained at 20° C. by the hanging drop method using aprotein solution of 5-10 mg/ml in 20 mM Tris pH 8.0, 100 mM NaCl, and2.5 mM MnCl₂ and a reservoir composition of 1.2 M Li₂SO₄, 100 mM MES pH6.0, 10 mM magnesium acetate and 3% ethylene glycol. Diffraction datawere collected on beamlines ID14-4 and ID23-1 at the EuropeanSynchrotron Radiation Facility (ESRF). The structure was solved by thesingle-wavelength anomalous dispersion (SAD) method using a gadoliniumchloride soaked crystal. Nine sites were found by SHELXD (Schneider andSheldrick, 2002, Acta Crastallogr. D. Biol. Crystallogr. 58:1772-1779)and refined with SHARP (de La

Fortelle et al., 1997, Methods in Enzymology 276:472-494). Afterthree-fold NCS averaging with RESOLVE (Terwilliger, 2002, ActaCrystallogr. D. Biol. Crystallogr. 58:2213-2215) an interpretable mapwas obtained and much of the model could be built with ARP/wARP(Perrakis et al., 1999, Nat. Struct. Biol. 6:458-463). Additionally,data were measured on a native crystal at the manganese K edge (X-raywavelength 1.89 Å) to reveal the location and identity of boundmanganese ions through anomalous difference Fourier synthesis. There arethree molecules in the asymmetric unit denoted A, B, and C. The metalion structure is best defined in molecule A. The crystallographicstatistics are summarized in Table 1 and more details available in theexperimental Examples below.

TABLE 1 Data collection and refinement statistics of PA-Nter PA-NterPA-Nter PA-Nter native Mn K-edge Gd derivative Data collection Beamline(ESRF) ID14-4 ID23-1 ID14-4 Wavelength (Å) 0.976 1.892 1.008 Space groupP4₃2₁2 P4₃2₁2 P4₃2₁2 Cell dimensions a, b, c (Å) 67.1, 67.1, 67.9, 67.9,67.8, 67.8, 302.9 300.8 300.4 α, β, γ (°) 90.0, 90.0, 90.0, 90.0, 90.0,106.24, 90.0 90.0 90.0 Resolution (Å) 50-2.05 30-2.60 30-2.5(2.05-2.10)* (2.6-2.7)* (2.5-2.6)* R_(merge)  0.056 (0.690) 0.055(0.484) 0.058 (0.539) l/σ/ 17.6 (2.2) 17.8 (2.5)  14.5 (2.1) Completeness (%)  93.2 (99.4) 99.7 (99.8) 97.9 (98.0) Redundancy  4.84(5.64) 3.66 (3.44) 3.63 (3.15) Refinement Resolution (Å) 30-2.05(2.05-2.10)* Total No. 39715/2118 reflections/free R_(work) 0.217(0.278) R_(free) 0.268 (0.320) No. atoms Protein 4742 Water/sulphate/Mnions 152/8/5 Average B-factors (Å²) All atoms 45.8 Chains A, B, D 41.5,40.0, 57.0 R.m.s. deviations Bond lengths (Å) 0.014 Bond angles (°)1.363 Ramachandran Plot** Favoured (%) 98.1 Allowed (%) 99.8

Example 1 Cloning, Expression and Purification

The DNA coding for PA residues 1-209 of the amino acid sequence setforth in SEQ ID NO: 2 (A/Victoria/3/1975 (H3N2)) was cloned into apET-M11 expression vector (EMBL) between the NcoI and XhoI sites. Apolypeptide linker having the amino acid sequence

GMGSGMA (SEQ ID NO: 19) was engineered after the tobacco etch virus(TEV) cleavage site to obtain a 100% cleavage by TEV protease. Thisvector was used to transform the BL21(DE3)-RIL-CodonPlus E. coli strain(Stratagene). The protein was expressed in LB medium overnight at 15° C.after induction with 0.1 mM isopropyl-β-thiogalactopyranoside (ITPG).The protein was purified by an immobilised metal affinity column (IMAC).A second IMAC step was performed after cleavage using a His-tagged TEVprotease, followed by gel filtration on a Superdex 200 column (GEHealthcare). Finally, the protein was concentrated to 5 to 10 mg/ml.

Example 2 Endonuclease Assay

All ribonucleic acid substrates for endonuclease assays were obtained byin vitro T7 transcription as described previously (Price et al., 1995,J. Mol. Biol. 249:398-408). Two structured RNAs were used: Alu-RNA; 110nucleotides comprising the Alu-domain of

Pyrococcus horikoshii signal recognition particle (SRP) RNA (unpublishedconstruct) and Candida albicans tRNA^(Asn) composed of 76 nucleotides(unpublished construct). We also used a uridine-rich unstructured RNA of51 nucleotides (U-rich RNA; 5′-GGCCAUCCUGU₇CCCU₁₁CU₁₉-3′; SEQ ID NO: 18)(Saito et al., 2008, Nature 454:523-527) and two partially folded RNAsderived from influenza A virus genomic RNA segment 5: a panhandle RNA(ph-RNA) of 81 nucleotides (Baudin et al., 1994, EMBO J. 13:3158-3165)and a shorter panhandle RNA (short ph-RNA) of 36 nucleotides comprisingjust the conserved 3′- and 5′-ends with a short linker (unpublishedconstruct). The endonuclease activity was also tested using a circularsingle stranded DNA (M13mpl 8) (Fermentas). RNA cleavage was performedby incubating 13 μM PA-Nter with various RNA substrates (all at 10 μM)at 37° C. in a final volume of 50 μL. The reaction buffer was 20 mMTris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol, and 1 mM metalsalts. Incubations were stopped by addition of EGTA at a finalconcentration of 20 mM. The reaction products were loaded on 8 M ureapolyacrylamide gels (8% or 15%) and stained with methylene blue. Theeffect of divalent cations on the RNAse activity of PA-Nter was testedat pH 8 (with β-mercaptoethanol) and pH 7 (without β-mercaptoethanol) byincubating ph-RNA with PA-Nter in the presence of different metal salts:MnCl₂, CaCl₂, MgCl₂, ZnCl₂ (or NiCl₂ at pH 7) and CoCl₂. For DNAcleavage, circular single stranded M13mp18 DNA was used. In the 10 μL,reaction volume (same buffer as for RNA), 100 ng/μL of purified plasmidMl3mp18 was incubated for 60 minutes in the presence of PA-Nter and 1 mMMnCl₂. The reaction products were loaded on a 0.8% agarose gel andstained with ethidium bromide. For endonuclease inhibition by2,4-Dioxo-4-phenylbutanoic acid (DPBA), PA-Nter and ph-RNA or singlestranded M13mp18 DNA were incubated in the presence of 1 mM MnCl₂ andincreasing concentrations of DPBA. Because DPBA is poorly soluble inwater, a stock solution of 65 mM DPBA was prepared in 50% ethanol thatwas further diluted so that only 1 4, of DPBA solution had to be addedto each reaction mix to obtain the required final concentration.Addition of the inhibitor in ethanol did not change the pH of thereaction mixture and the addition of the same concentration of ethanolalone had no effect on nuclease activity (not shown).

Using a partially structured 81nt ph-RNA it could be demonstrated thatPA-Nter has intrinsic RNase activity that is divalent cation dependent(FIG. 5). Consistent with the results on RNPs (Doan et al., 1999,Biochemistry 38:5612-5619, strong activity was observed at pH 8 withmanganese and weaker activity with magnesium ions. At pH 7, the PA-Nterendonuclease activity was also observed with cobalt (FIG. 6). After 40minutes incubation highly structured RNAs such as tRNA and SRP Alu-RNAwere relatively resistant to degradation, partially structured ph- andshort-ph-RNAs were partially degraded and unstructured U-rich RNA wascompletely degraded, suggesting that the enzyme is single-strandspecific (FIG. 7). The enzyme also completely degraded circular ssDNAshowing that it is a nonspecific endonuclease (FIG. 8). The endonucleaseactivity on both RNA and

DNA was inhibited in a dose dependent manner by the compound2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of influenzaendonuclease (FIG. 9). The K, for this compound is estimated at 26 μM,in excellent agreement with the IC₅₀ reported for the same compoundinhibiting cleavage of capped RNA by the intact influenza viruspolymerase (Tomassini et al., 1994, Antimicrob. Agents Chemother38:2827-2837).

Example 3 Thermal Shift Assay

Thermal shift assays were performed with 10 μM of PA-Nter in 20 mMTris-HC1 pH 7.0 or 8.0, 100 mM NaC1 and a 5X dilution of SYPRO Orangedye (Invitrogen) as described (Ericsson et al., 2006, Anal. Biochem.357:289-298). The dye was excited at 490 nm and the emission light wasrecorded at 575 nm while the temperature was increased by increments of1° C. per minute from 25 to 75° C. Control assays were carried out inthe absence of protein or dye to check that no fluorescence signal wasrecorded.

The thermal shift assay was performed to investigate the thermalstability of PA-Nter in presence and absence of divalent cations. Theexperiments revealed a significant increase in thermal stability(apparent melting temperature shifts from 44° C. to 57° C.) uponaddition of manganese ions and to a lesser extent upon addition ofcalcium and magnesium ions (FIGS. 1 and 2).Titrating the compound2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of influenzaendonuclease, to manganese bound PA-Nter increases the thermal stabilityeven further (apparent melting temperature shifts form 59° C. to 65° C.)(FIG. 4), whereas the inhibitor has no effect on metal-free enzyme (datanot shown).

Example 4 Far UV Circular Dichroism (CD) Spectroscopy

Far-UV CD spectra were recorded with 1 mM path length at 20° C. on aJASCO model

J-810 CD spectro-polarimeter equipped with a Peltier thermostat. ThePA-Nter concentration was 10 μM in 10 mM Tris-HC1, pH 8.0, 10 mM NaC1 inthe presence or absence of 1 mM MnCl₂. Mean residue ellipticity wascalculated using the number of residues (PA-Nter is 209 residues longplus 7 additional residues before the starting methionine). Wavelengthscans were recorded from 200 to 260 nm and averaged over eightconsecutive scans (0.5 nm increment, 1 s response, 1 nm bandwidth and 50nm/min scanning speed).

The structural effect of manganese binding to PA-Nter, investigated byCD spectroscopy, revealed a significant increase in helical content(estimated 8 to 9 residues) upon addition of 1 mM Mn²⁺ (FIG. 3).

Example 5 Crystallization and Crystallography

Initial sitting drop screening was carried out at 20° C. mixing 100 nLof protein solution (6 mg/ml) with 100 nL of well solution using aCartesian robot. Subsequently, larger crystals were obtained at 20° C.by the hanging drop method following a ratio of 1:1 well:proteinsolutions. The protein solution was at 5-10 mg/ml in 20 mM Tris-HC1 pH8.0, 100 mM NaC1, 2.5 mM MnCl₂. The reservoir composition was 100 mM MESpH 6.0, 1.2 M Li₂SO₄, 10 mM magnesium acetate, 3% ethylene glycol afterrefinement of the crystallisation condition. Crystals appeared after 1-2weeks and were typically of a volume of 50×50×15 μm³.

Crystals were frozen in liquid nitrogen in the presence of 22% ethyleneglycol for cryoprotection. Diffraction data were collected at 100 K onbeamlines ID14-4 and ID23-1 at the European Synchrotron RadiationFacility (ESRF) and all data were integrated and scaled in the spacegroup P4₃2₁2 using the XDS suite (Kabsch, 1993, J. Appl. Cryst.26:795-800). The best native data were collected to 2.05 Å resolution ata wavelength of 0.976 Å, after soaking with additional 10 mM MnCl₂ for 2minutes. Additionally, data was measured on native crystals at awavelength of 1.89 Å (close to the manganese K edge) to reveal thelocation and identity of any bound manganese ions. The structure wassolved with a highly redundant data set to 2.5 Å resolution collected ata wavelength of 1.008 Å from a crystal soaked for 6 h in mother liquorcontaining 5 mM GdCl₃. Three initial Gd sites were located on the basisof their anomalous differences using SHELXD (Schneider and Sheldrick,2002, Acta Crystallogr. D. Biol. Crystallogr. 58:1772-1779) asimplemented in HKL2MAP (Pape and Schneider, 2004, J. Appl. Cryst.37:843-844). These initial sites were refined and experimental phases to3.5 Å were calculated using the single anomalous dispersion (SAD)procedure in SHARP (de La Fortelle et al., 1997, Methods in Enzymology276:472-494). After several iterative cycles a further 6 sites wereidentified in the residual maps and the phases were refined to 2.5 Å.These initial phases were improved with the density modification packageSOLOMON in SHARP. Finally, a clearly interpretable map was obtained byusing 3-fold NCS operators identified from the 9 Gd sites by RESOLVE(Terwilliger, 2002, Acta Crystallogr. D. Biol. Crystallogr.58:2213-2215) for averaging with DM (Cowtan, 1994, Joint CCP4 andESF-EACBM Newsletter on Protein Crystallography 31:34-38) as implementedin CCP4 (Collaborative Computational Project, 1994, Acta Crystallogr. D.Biol. Crystallogr. 50:760-763). This averaged map was of sufficientquality for RESOLVE (Terwilliger, 2003, Acta Crystallogr. D. Biol.Crystallogr. 59:45-49) to build 396 out of 648 possible amino acids, ofwhich 85 could be sequence assigned. A manually modified model and asubsequent high resolution data set to 2.05 Å were then put into

ARP/wARP (Perrakis et al., 1999, Nat. Struct. Biol. 6:458-463) resultingin a more complete model. This model was refined with Refmac (Murshudov,1997, Acta Crystallogr. D. Biol. Crystallogr. 53:240-255) iterated withmanual rebuilding cycles in 0 (Jones et al., 1991, Acta Crystallogr. A47:110-119). Using TLS refinement and tight NCS restraints on parts ofthe structure, the final R-factor (R-free) is 0.233 (0.291). Accordingto MOLPROBITY (Lovell et al., 2003, Proteins 50:437-450), 97.5%, 99.8%are respectively in the favoured and allowed region of the Ramachandranplot. The crystallographic details are summarized in Table 1. There arethree molecules in the asymmetric unit denoted A, B, and D. The metalion structure is best defined in molecule A. Different molecules haveregions 69-74 and 134-143 more or less well ordered. 6 residues of theN-terminal tag and residues 204-209 are not visible. Molecule D is theleast well ordered overall (Table 1). In the described structure thecrystal contact between two of the molecules (B and D) exhibits multipleconformations perhaps accounting for the relatively high R-factor of thenative data for the resolution. Structure figures were drawn with PyMOL(DeLano, 2002, available online at http://www.pymol.sourceforge.net).The sequence alignment in FIG. 11 was drawn with

ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Gouet etal., 1999, Bioinformatics 15:305-308). The electrostatic surface (FIG.13) was calculated using DelPhi (Rocchia et al., 2002, J. Comput. Chem.23:128-137). Structural similarity searches were performed with MSDFOLD(http://www.ebi.ac.uk/msdsrv/ssm/cgi-bin/ssmserver) and Dalilite(http://www.ebi.ac.uk/Tools/dalilite/index.html).

We grew small square-plate crystals of PA-Nter in the presence of bothmanganese and magnesium that diffracted to about 2 Å resolution, withthree independent molecules in the asymmetric unit. The crystalstructure reveals a single, folded domain with residues 1-196 visible,comprising seven α-helices and a mixed, five-stranded β-sheet (FIG. 10).The structure based sequence alignment amongst influenza A, B and Cviruses (FIG. 11) projected onto a surface representation reveals a veryhighly conserved depression that is strongly negatively charged due to aconcentration of acidic residues (FIGS. 12 and 13), suggestive of anactive site. A structure similarity search gave no high scoring hitsindicating that the global fold is novel. The most similar protein foundis the archaeal Holliday junction resolvase Hjc from Pyrococcus furiosus(Nishino et al., 2001, Structure 9:197-204). The structural alignment ofPA-Nter with Hjc superposes helix α3 and strands β1-5 (FIG. 14, left andmiddle panel) encompassing a structural motif characteristic of manynucleases including resolvases and type II restriction enzymes. Themotif includes catalytically important divalent metal ion binding acidicresidues Asp33 and Glu46 of Hjc upon which Asp108 and Glu119 of PA-Nterexactly superpose. Structural alignment of PA-Nter with type IIrestriction endonucleases such as BamHI or EcoRV reveals a similarsuperposition of active site elements (FIG. 14, right panel).Catalytically important Glu45, Asp74, Asp90 and Lys92 of EcoRV alignwith His41, Asp108, Glu119 and Lys134 of PA-Nter, respectively, althoughthe lysines are positioned differently in the primary sequence (FIG.16). The conserved lysine is implicated in stabilizing the attackinghydroxide nucleophile during catalysis. Thus PA-Nter is a new member ofthe PD-(D/E)XK nuclease superfamily which encompasses a diversity ofenzymes involved in various aspects of DNA metabolism. In PA-Nter, thecharacteristic motif occurs at 107-PDLYDYK (SEQ ID NO: 20), although theseparation between the two acidic residues is unusually short and theputative catalytically important lysine (Lys 134) has ‘migrated’ to analternative position, as in some other members of the superfamily.Within this family, PA-Nter is unusual in that it is biologicallyfunctional as an RNase and has a histidine in the active site.

To confirm that the conserved acidic residues of PA-Nter are metalbinding residues we calculated an anomalous difference map using datacollected at the manganese K absorption edge. Two manganese ions wereidentified in each active site as adjacent anomalous peaks separated byabout 3.8 Å (FIG. 15, left panel). The stronger peak (Mn1) isco-ordinated by Glu80, Asp108 and two water molecules; the weaker site(Mn2) by His41, Asp108, Glu119 and the carbonyl oxygen of Ile120. Thecited residues are absolutely conserved in all influenza virus PAsequences (except for Ile120 which is conservatively substituted) (FIG.11). The two metal sites correspond closely with those observed inrestriction enzymes such as EcoRV (FIG. 15, right panel). His41(positioned as Glu45 in EcoRV) from helix α3 could be important inconferring manganese specificity, since magnesium and calcium bind lessreadily to histidine. Manganese binding by His41 and the resultingstabilization of helix α3 could account for the additional helicalcontent (estimated as 8-9 residues) detected upon incubating PA-Nterwith manganese (FIG. 3). In the crystal, Mn1 is also co-ordinated byGlu59 from a loop of an adjacent molecule. Superposition of DNAcomplexes of BamHI or EcoRV on PA-Nter shows that the Glu59 carboxylategroup corresponds closely to the position of the scissile phosphategroup (FIG. 17). Thus our structure mimics a substrate or productcomplex.

Our structural and biochemical results combined with previousobservations on the trimeric polymerase provide compelling evidence thatPA-Nter is the endonuclease that cleaves host mRNAs duringcap-snatching. First, the domain has intrinsic RNA and DNA endonucleaseactivity which is preferentially activated by manganese, in accordancewith observations reported for the viral RNPs (FIG. 6). Second, thisactivity is inhibited by a compound known to inhibit influenzaendonuclease activity with a nearly identical K_(i) (FIG. 9). Third, thedomain contains a structural motif characteristic of the catalytic coreof a broad family of nucleases, including type II endonucleases. Theactive site features a cluster of three acidic residues (Glu80, Asp108and Glu119) and a putative catalytic lysine (Lys134) (FIGS. 14 to 16).Fourth, these acidic residues, together with His41, are all absolutelyconserved in influenza viruses, co-ordinate two manganese ions in aconfiguration consistent with a two-metal dependent reaction mechanismas proposed for many nucleases (FIG. 15, left panel).

1. A polypeptide fragment comprising an amino-terminal fragment of thePA subunit of a viral RNA-dependent RNA polymerase possessingendonuclease activity, wherein said PA subunit is from a virus belongingto the Orthomyxoviridae family.
 2. The polypeptide fragment of claim 1,which is soluble.
 3. The polypeptide fragment of claim 1, which iscrystallizable.
 4. The polypeptide fragment of claim 3 which iscrystallizable using a protein solution of 5 to 10 mg/ml in 20 mM TrispH 8.0, 100 mM NaCl and 2.5 mM MnCl₂ and a reservoir solution consistingof 1.2 M Li₂SO₄, 100 mM MES pH 6.0, 10 mM magnesium acetate, and 3%ethylene glycol.
 5. The polypeptide fragment of claim 1, wherein the PAsubunit is from Influenza A, B, or C virus or is a variant thereof. 6.The polypeptide fragment of claim 1, wherein the amino terminal fragmentcorresponds to at least amino acids 1 to 196 of the PA subunit of theRNA-dependent RNA polymerase of Influenza A virus (SEQ ID NO: 2).
 7. Thepolypeptide fragment of claim 1, wherein said polypeptide fragment ispurified to an extent to be suitable for crystallization.
 8. Thepolypeptide fragment of claim 1, which two divalent cations are bound,wherein the divalent cation is preferably manganese.
 9. The polypeptidefragment of claim 1, wherein (i) the N-terminus is identical to orcorresponds to amino acid position 1 and the C-terminus is identical toor corresponds to an amino acid at a position selected from positions196 to 209 of the amino acid sequence of the PA subunit according to SEQID NO: 2, (ii) the N-terminus is identical to or corresponds to aminoacid position 1 and the C-terminus is identical to or corresponds to anamino acid at a position selected from positions 195 to 206 of the aminoacid sequence of the PA subunit according to SEQ ID NO: 4, or (iii)wherein the N-terminus is identical to or corresponds to amino acidposition 1 and the C-terminus is identical to or corresponds to an aminoacid at a position selected from positions 178 to 189 of the amino acidsequence of the PA subunit according to SEQ ID NO: 6, and variantsthereof, which retain the endonuclease activity.
 10. The polypeptidefragment of claim 1 which consists of amino acids 1 to 209 of the aminoacid sequence set forth in SEQ ID NO: 2 and optionally an amino-terminallinker having the amino acid sequence GMGSGMA (SEQ ID NO: 19) and whichhas the structure defined by the structure coordinates as shown in FIG.18.
 11. The polypeptide fragment of claim 1, wherein said polypeptidefragment has a crystalline form with space group P4₃2₁2 and unit celldimensions of a=b=6.71±0.2 nm, c=30.29 nm±0.4 nm.
 12. The polypeptidefragment of claim 11, wherein the crystal diffracts X-rays to aresolution of 2.5 Å or higher, preferably 2.1 Å or higher.
 13. Anisolated polynucleotide coding for an isolated polypeptide of claim 1.14. A recombinant vector comprising said isolated polynucleotide ofclaim
 13. 15. A recombinant host cell comprising said isolatedpolynucleotide of claim
 13. 16. A method for identifying compounds whichmodulate the endonuclease activity of the PA subunit of a viralRNA-dependent RNA polymerase from the Orthomyxoviridae family,comprising the steps of (a) constructing a computer model of the activesite defined by the structure coordinates of the polypeptide fragment ofclaim 1 which consists of amino acids 1 to 209 of the amino acidsequence set forth in SEQ ID NO: 2 and optionally an amino-terminallinker having the amino acid sequence GMGSGMA (SEQ ID NO: 19) and whichhas the structure defined by the structure coordinates as shown in FIG.18; (b) selecting a potential modulating compound by a method selectedfrom the group consisting of: (i) assembling molecular fragments intosaid compound, (ii) selecting a compound from a small molecule database,and (iii) de novo ligand design of said compound; (c) employingcomputational means to perform a fitting program operation betweencomputer models of the said compound and the said active site in orderto provide an energy-minimized configuration of the said compound in theactive site; and (d) evaluating the results of said fitting operation toquantify the association between the said compound and the active sitemodel, whereby evaluating the ability of said compound to associate withthe said active site.
 17. The method of claim 16, wherein said activesite comprises amino acids corresponding to amino acids Asp108, Ile120,and Lys134 of the PA subunit according to SEQ ID NO:
 2. 18. The methodof claim 17, wherein said active site further comprises amino acidscorresponding to amino acids His41, Glu80, and Glu119 of the PA subunitaccording to SEQ ID NO:
 2. 19. The method of claim 17, wherein saidactive site further comprises the amino acids corresponding to aminoacids Tyr24, Arg84, Leu106, Tyr130, Glu133, and Lys137 of the PA subunitaccording to SEQ ID NO:
 2. 20. The method of claim 16, wherein saidactive site is defined by the structure coordinates of the PA subunitSEQ ID NO: 2 amino acids Asp108, Ile120, and Lys134 according to FIG.18.
 21. The method of claim 20, wherein said active site is furtherdefined by the structure coordinates of the PA subunit SEQ ID NO: 2amino acids His41, Glu80, and Glul 19 according to FIG.
 18. 22. Themethod of claim 20 or 21, wherein said active site is further defined bythe structure coordinates of the PA subunit SEQ ID NO: 2 amino acidsTyr24, Arg84, Leu106, Tyr130, Glu133, and Lys137 according to FIG. 18.23. The method of claim 21, wherein the active site of a PA subunitpolypeptide fragment variant has a root mean square deviation from thebackbone atoms of the amino acids Asp108, Ile120, and Lys134 of saidactive site of not more than 2.5 Å.
 24. The method of any of claims 16comprising the further step of (e) synthesizing said compound andoptionally formulating said compound or a pharmaceutically acceptablesalt thereof with one or more pharmaceutically acceptable excipient(s)and/or carrier(s).
 25. The method of claim 24 comprising the furtherstep of (f) contacting said compound and said polypeptide fragment ofclaim 1 and determining the ability of said compound to modulate theendonuclease activity of said PA subunit polypeptide fragment.
 26. Acompound identifiable by the method of claim 16, wherein said compoundis able to modulate the endonuclease activity of the PA subunit orvariant thereof.
 27. A compound identifiable by the method of claims 16,wherein said compound is able to inhibit the endonuclease activity ofthe PA subunit polypeptide fragment of a polypeptide fragment comprisingan amino-terminal fragment of the PA subunit of a viral RNA-dependentRNA polymerase possessing endonuclease activity, wherein said PA subunitis from a virus belonging to the Orthomyxoviridae family, or variantthereof.
 28. A method for identifying compounds which modulate theendonuclease activity of the PA subunit or polypeptide variants thereof,comprising the steps of (i) contacting said polypeptide fragment ofclaims 1 with a test compound and (ii) analyzing the ability of saidtest compound to modulate the endonuclease activity of said PA subunitpolypeptide fragment.
 29. The method of claim 28, wherein the ability ofsaid test compound to inhibit the endonuclease activity of said PAsubunit polypeptide fragment is analyzed.
 30. The method of claim 28performed in a high-throughput setting.
 31. The method of any of claim28, wherein said test compound is a small molecule.
 32. The method ofany of claims 28, wherein said test compound is a peptide or protein.33. The method of any claim 16, wherein said method further comprisesthe step of formulating said compound or a pharmaceutically acceptablesalt thereof with one or more pharmaceutically acceptable excipient(s)and/or carrier(s).
 34. A pharmaceutical composition producible accordingto the method of claim
 24. 35. A compound identifiable by the method ofclaim 28, wherein said compound is able to modulate the endonucleaseactivity of the PA subunit or variant thereof.
 36. A compoundidentifiable by the method of claim 28, wherein said compound is able toinhibit the endonuclease activity of the PA subunit polypeptide fragmentof a polypeptide fragment comprising an amino-terminal fragment of thePA subunit of a viral RNA-dependent RNA polymerase possessingendonuclease activity, wherein said PA subunit is from a virus belongingto the Orthomyxoviridae family, or a variant thereof.
 37. An antibodydirected against the active site of the PA subunit or variant thereof.38. The antibody of claim 37, wherein said antibody recognizes apolypeptide fragment of a length between 5 and 15 amino acids of theamino acid sequence as set forth in SEQ ID NO: 2, wherein thepolypeptide fragment comprises one or more amino acid residues selectedfrom the group consisting of Tyr24, His41, Glu80, Arg84, Leu106, Asp108,Glu119, Ile120, Tyr130, Glu133, Lys134, and Lys137.
 39. Use of acompound according to claim 26, or a pharmaceutical composition thereoffor the manufacture of a medicament for treating, ameliorating, orpreventing disease conditions caused by viral infections with viruses ofthe Orthomyxoviridae family.
 40. The use of claim 39, wherein saiddisease condition is caused by a virus selected from the groupconsisting of Influenza A virus, Influenza B virus, and Influenza Cvirus.
 41. The use of an antibody according to claim 37 for themanufacture of a medicament for treating, ameliorating, or preventingdisease conditions caused by viral infections with viruses of theOrthomyxoviridae family.
 42. The use of the antibody according to claim41, wherein said disease condition is caused by a virus selected fromthe group consisting of Influenza A virus, Influenza B virus andInfluenza C virus.
 43. The method of claim 28, wherein said methodfurther comprises the step of formulating said compound or apharmaceutically acceptable salt thereof with one or morepharmaceutically acceptable excipient(s) and/or carrier(s).
 44. Apharmaceutical composition producible according to the method of claim33.
 45. A compound identifiable by the method of claim 33, wherein saidcompound is able to modulate the endonuclease activity of the PA subunitor variant thereof.
 46. A compound identifiable by the method of claim33, wherein said compound is able to inhibit the endonuclease activityof the PA subunit polypeptide fragment of a polypeptide fragmentcomprising an amino-terminal fragment of the PA subunit of a viralRNA-dependent RNA polymerase possessing endonuclease activity, whereinsaid PA subunit is from a virus belonging to the Orthomyxoviridaefamily, or a variant thereof.
 47. Use of a compound according to claim27 or a pharmaceutical composition thereof for the manufacture of amedicament for treating, ameliorating, or preventing disease conditionscaused by viral infections with viruses of the Orthomyxoviridae family.48. Use of a compound according to claim 35, or a pharmaceuticalcomposition thereof for the manufacture of a medicament for treating,ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family.
 49. Use of acompound according to claim 36, or a pharmaceutical composition thereoffor the manufacture of a medicament for treating, ameliorating, orpreventing disease conditions caused by viral infections with viruses ofthe Orthomyxoviridae family.
 50. Use of a pharmaceutical compositionaccording to claim 34, for the manufacture of a medicament for treating,ameliorating, or preventing disease conditions caused by viralinfections with viruses of the Orthomyxoviridae family.